Compositions and methods for controlling head blight disease

ABSTRACT

Compositions comprising microbiological strains and cultures and methods of use thereof are provided herein. Certain strains, cultures, and compositions thereof are useful for the control of head blight disease, for example, of various crop plants. Biological control compositions, and methods of use thereof to prevent, inhibit or treat the development of plant pathogens or disease and for preserving plant yield, are also provided.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. § 119(e) to U.S.Provisional Patent Application No. 61/511,467, filed on Jul. 25, 2011,the entire contents of which is herein incorporated by reference.

INCORPORATION OF SEQUENCE LISTING

The material in the accompanying Sequence Listing is hereby incorporatedby reference in its entirety. The accompanying file, named“SGI1520-1_ST25.txt”, was created on Jul. 24, 2012 and is 52 Kb. Thefile can be accessed using Microsoft Word on a computer that uses WindowOS.

FIELD OF THE INVENTION

The present invention relates to the biological control ofphytopathogenic diseases. Specifically, it relates to compositions andmethods useful for controlling head blight disease in cereal plants,such as wheat and barley.

BACKGROUND OF THE INVENTION

Head blight, also known as head scab, pink mold, whiteheads andtombstone scab, is a devastating disease afflicting wheat, barley, andseveral other cereal crops worldwide, particularly in USA, Europe andChina. This disease can reach epidemic levels and cause extensive damageto grains, especially wheat and barley in humid and semi-humid cerealgrowing areas of the world, including India, Russia, France, Germany,and the United Kingdom. Particularly, wheat scab or head blight is oneof the more damaging diseases of wheat in the United States. Nationwide,this disease has caused the wheat industry millions of dollars in yieldlosses. In the Midwest and High Plains, wheat scab is the major obstacleto wheat production in recent years. Head blight in addition toattacking wheat also attacks and reproduces on barley, oats, corn, andmany other cereals.

This serious plant disease can be caused by several phytopathogens, butprimarily by several species in the fungal genus Fusarium. For example,causal agents of the head blight disease in wheat include a number ofdifferent Fusarium species, e.g. F. culmorum, F graminearum (teleomorph,Gibberella zeae), F. avenaceum (teleomorph, G. avenacea), F. poae, aswell as by non-Fusarium pathogens such as Microdochium nivale(teleomorph, Monographella nivalis), and Microdochium majus. In theUnited States, Europe and most agronomically important areas of theworld, the predominant causative agent of head blight is Fusariumgraminearum (teleomorph, Gibberella zeae sensu strict).

These pathogens typically survive on plant debris. They invade anddamage the spikelets of the grain head during flowering, thus preventingor partially impeding the development of grain in the grain head. As aresult, the invading scab pathogen can either kill part of the grainhead or the entire grain head. Some infected seeds are low in vigor andoften fail to germinate. Infected seeds that germinate often die earlyin the seedling stage due to crown rot or root rot, causing poor standsin the following crop plant. Healthy seedlings can also become infectedat emergence. In addition to poor, unthrifty stand, yield losses due topathogen infestation can be quite high if conditions are favorable fordevelopment of the disease.

The fungal pathogens of Fusarium genus spread across grain cultivatingareas all over the world, and are known to cause severe damage,particularly in areas with high rainfall between flowering and grainfilling. When Fusarium graminearum is the causative agent, this diseaseis of primary concern because it does not only reduce the commercialvalues of the contaminated grains, in addition to yield losses, butbecause Fusarium infection can also lead to the accumulation oftrichothecene mycotoxins in the grains thus threatening the health ofhuman and livestock. Trichothecenes are major mycotoxin contaminants ofcereals worldwide, causing feed refusal, vomiting, diarrhea and weightloss in non-ruminant animals and posing a health threat to other animalsand humans when exposure levels are high. This threat has beenexacerbated by the recent shift in the F. graminearum strains in theUnited States towards greater toxin production and vigor. Mostfrequently found mycotoxins are deoxynivalenol (DON, also known asvomitoxin) and zearalenone (ZEA). Deoxynivalenol in particular is a verydangerous toxin, causing gastrointestinal disorders accompanied byhemorrhagic conditions and the like in humans and animals that eatinfected grains, leading to death in some cases. Since deoxynivalenol isgenerally stable against changes in pH and high temperature,detoxification can be very difficult. Therefore, grains contaminatedbeyond a certain level cannot be used in any form of brewing,processing, or livestock feed, and thus often need to be disposed of.

To date, various strategies have been deployed for controlling headblight in crop plants. Promising options include chemical measures, thedevelopment of resistant crop cultivars, and traditional practices ofcrop rotation and tillage of fields. Among these options, chemicalpesticides can be somewhat effective in reducing head blightinfestation, but residue concerns regarding the use of fungicides latein crop development, typically at flowering stages only a few weeksbefore harvest, lessen their attractiveness. Advances in developingresistant cultivars using traditional breeding and genetic engineeringrepresent another disease control alternative are also occurring.Reported examples of genetic engineering advances include altering theproduction of a plant hormone or manipulating the plant hormonesignaling pathway. In recent years, considerable advances in the area oftraditional breeding have been made in understanding the genetic basisof resistance to head blight disease and a number of genes andquantitative trait loci (QTL) conferring resistance have been reported.However, progress in improving crop resistance to head blight diseasehas been slow, largely because of the difficulty of studying thisdisease. In fact, relatively little is currently understood about themechanisms involved in resistance or susceptibility. In addition, thegenetic diversity of Fusarium species, which are the predominantcausative agents of the disease, often raises concerns regarding howdurable the efficacy of chemical fungicides and resistant cultivars willbe. As a result, practically all wheat cultivars currently inlarge-scale production remain vulnerable to infection.

In addition, although some success in controlling head blight diseasecan be expected by traditional practices such as plowing fields to burycrop residues infested with causative agent, e.g. F. graminearum, afterharvest, conventional tillage of fields after harvesting is notcompatible with the soil conservation practice of minimum tillage.Considering the potential of long distance inoculum dispersal and thediverse crops that can act as alternative hosts of the pathogens, croprotation is often an untenable solution. In addition to the problem ofpesticide residues in the environment, reports of pesticide resistanceand instances of DON content increases in grain can also be concernswith their use. Further, costs and increasing concerns in the public andprivate sectors over pesticide residues in the environment and foodproduct safety render this disease control alternative less attractive,and have led to requests for crop cultivation using as little pesticidesas possible.

In summary, despite considerable advances in developing techniques tocontrol head blight, reducing the impact of this devastating disease ongrain production and quality remains an intractable problem. Therefore,identification and development of new head blight controlling techniquesis essential in improving the production and quality of many cerealcrops. These problems require urgent solution not only in United States,but also across the globe, including the Asia and Europe.

Biological control of head blight disease has attracted considerableinterest since the mid 1990's. Biological control agents (BCAs), thoughcurrently very limited in number, could be an environmentally acceptablemethod for substantially decreasing the level of disease incited bypathogens such as Fusarium. Public acceptance, compatibility with otherdisease management measures, and durability are among favorable factorsin support of developing strategies for biologically controlling headblight disease. Biological control agents could play an important rolein organic cereal production. In conventional production, such agentsmay extend protection of spikes past the flowering stage after chemicalfungicides can no longer be applied. To date, significant advances inthe area of biological control have been achieved. For example, certainstrains of spore-producing bacteria (such as Bacillus and Pseudomonasspecies) and yeasts (such as Cryptococcus species) show some promise forthe control of head blight disease and the reduction of mycotoxincontamination. However, despite these and other advances, the needremains for improved microorganisms for use in the biological control ofhead blight disease. Although BCAs have become a more acceptablesolution for plant pathogens and BCA products have been marketed to agreater extent than ever before, to date there have been few attempts todevelop strategies and antagonistic microorganism for biologicallycontrolling head blight disease. Furthermore, the life cycle of Fusariumspp. and other causative agents of head blight disease suggest that thepathogens can potentially be susceptible to biocontrol techniques usingantagonistic microorganisms at different developmental stages. Thus,there is a need to identify new biological control agents, preferablywith different modes of actions, as well as biocontrol methods that canhelp effectively prevent or suppress the development of head blightdisease.

SUMMARY OF THE INVENTION

Compositions comprising microbiological strains and cultures areprovided herein. Certain strains, cultures, and compositions thereof areuseful for the control of head blight disease, for example, of variouscrop plants including wheat and other cereal plants. Biological controlcompositions, and methods of use thereof to prevent, inhibit or treatthe development of plant pathogens or disease and for preserving plantyield, are also provided. Also provided are methods for the use of suchcompositions as biological control agents in combination with otheragriculturally effective compounds or compositions for controllingharmful plant pathogens.

In one aspect, the present invention provides isolated microbial strainshaving suppressive activity against head blight disease. The microbialstrains in accordance to this aspect of the present invention areselected from the group consisting of the genera Microbacterium,Bacillus, Mycosphaerella, and Variovorax. In some preferred embodiments,the microbial strains are selected from the group consisting ofMycosphaerella sp. strain SGI-010-H11 (deposited as NRRL 50471),Microbacterium sp. strain SGI-014-006 (deposited as NRRL B-50470),Microbacterium sp. strain SGI-005-G08 (deposited as NRRL _-______),Variovorax sp. strain SGI-014-G01 (deposited as NRRL B-50469), Bacillussp. strain SGI-015-F03 (deposited as NRRL B-50760), Bacillus sp. strainSGI-015-H06 (deposited as NRRL B-50761), and pesticidally activevariants of any thereof. The microbial strain in accordance to someother preferred embodiments can comprise a DNA sequence that exhibits atleast 85% sequence identity to any one of the nucleotide sequences inthe Sequence Listing. Further provided are biologically pure culturesand enriched cultures of the microbial strains disclosed herein.

Also provided in another aspect of the present invention arecompositions that comprise a microbial strain of the invention or aculture thereof, and an agriculturally effective amount of a compound orcomposition selected from the group consisting of an acaricide, abactericide, a fungicide, an insecticide, a microbicide, a nematicide, apesticide, and a fertilizer. The compositions in some embodiments ofthis aspect may be prepared as a formulation selected from the groupconsisting of an emulsion, a colloid, a dust, a granule, a pellet, apowder, a spray, an emulsion, and a solution. In some other embodiments,the compositions may be provided with a carrier. In some preferredembodiment, the carrier is an agriculturally acceptable carrier. In someparticularly preferred embodiment, the carrier is a plant seed. In otherpreferred embodiments, the composition is a seed coating formulation.Further provided in the present disclosure are seeds that are coatedwith a composition according to the present invention.

In another aspect of the present invention, there are provided methodsfor preventing, inhibiting or treating the development of a plantpathogen. The methods involve growing a microbial strain of theinvention or a culture thereof in a growth medium or soil of a hostplant prior to or concurrent with host plant growth in the growth mediumor soil. In some preferred embodiments of this aspect, the plantpathogen causes head blight disease. In some particularly preferredembodiments, the plant pathogen is Fusarium graminearum.

In yet another aspect of the present invention, methods are provided forpreventing, inhibiting or treating the development of head blightdisease of a plant. The methods involve applying to the plant, or to theplant's surroundings, an effective amount of a microbial strain of theinvention or a culture thereof. In one embodiment, such head blightdisease is caused by the fungus Fusarium graminearum. In someembodiments of this aspect, the microbial strain or a culture thereof isapplied to soil, a seed, a root, a flower, a leaf, a portion of theplant, or the whole plant. In a preferred embodiment, the plant issusceptible to Fusarium graminearum. In some other preferredembodiments, the plant is wheat plant, a corn plant, a barley plant, oran oat plant. In another preferred embodiment, the microbial strain ofthe present invention or a culture thereof is established as anendophyte on the plant.

Another further aspect of the invention provides non-naturally occurringplants. The non-naturally occurring plants are artificially infectedwith a microbial strain of the invention or a culture thereof. Furtherprovided in some preferred embodiments of this aspect are seed,reproductive tissue, vegetative tissue, plant parts, and progeny of thenon-naturally occurring plants.

Yet another aspect of the invention provides a method for preparing anagricultural composition. The method involves inoculating the microbialstrain according to the present invention or a culture thereof into oronto a substratum and allowing it to grow at a temperature of 1-37° C.until obtaining a number of cells or spores of at least 10²-10³ permilliliter or per gram.

These and other objects and features of the invention will become morefully apparent from the following detailed description of the inventionand the claims.

DETAILED DESCRIPTION OF THE INVENTION Some Definitions

Unless otherwise defined, all terms of art, notations and otherscientific terms or terminology used herein are intended to have themeanings commonly understood by those of skill in the art to which thisinvention pertains. In some cases, terms with commonly understoodmeanings are defined herein for clarity and/or for ready reference, andthe inclusion of such definitions herein should not necessarily beconstrued to represent a substantial difference over what is generallyunderstood in the art. Many of the techniques and procedures describedor referenced herein are well understood and commonly employed usingconventional methodology by those skilled in the art.

The singular form “a”, “an”, and “the” include plural references unlessthe context clearly dictates otherwise. For example, the term “a cell”includes one or more cells, including mixtures thereof.

The expressions “antagonistic microorganism” and “microbial antagonist”are used herein interchangeably in reference to a microorganism isintended to mean that the subject strain exhibits a degree of inhibitionof head blight disease exceeding, at a statistically significant level,that of an untreated control.

Antibiotic: the term “antibiotic”, as used herein, refers to anysubstance that is able to kill or inhibit the growth of a microorganism.Antibiotics may be produced by any one or more of the following: 1) amicroorganism, 2) a synthetic process, or 3) a semisynthetic process. Anantibiotic may be a microorganism that secretes a volatile organiccompound. Furthermore, an antibiotic may be a volatile organic compoundsecreted by a microorganism.

Bactericidal: the term “bactericidal”, as used herein, refers to theability of a composition or substance to increase mortality or inhibitthe growth rate of bacteria. Inhibition of the growth rate of bacteriacan be commonly quantified as the reduction of viable bacterial cellsover time.

Biological control: the term “biological control” and its abbreviatedform “biocontrol”, as used herein, is defined as control of a pathogenor insect or any other undesirable organism by the use of at least asecond organism other than man. An example of a known mechanism ofbiological control is the use of microorganisms that control root rot byout-competing fungi for space on the surface of the root, ormicroorganisms that either inhibit the growth of or kill the pathogen.The “host plant” in the context of biological control is the plant thatis susceptible to disease caused by the pathogen. In the context ofisolation of an organism, such as a fungal species, from its naturalenvironment, the “host plant” is a plant that supports the growth of thefungus, for example, a plant of a species the fungus is an endophyte of.

The term “cereal” as used herein is intended to refer to any cerealspecies that can be susceptible to head blight disease. Cereals reportedto be susceptible include wheat, barley, oats, and triticale, thoughwheat and barley are the two crops in which this disease presents asignificant economic problem.

An “effective amount” refers to an amount sufficient to affectbeneficial or desired results. In terms of disease management,treatment, inhibition or protection, an effective amount is that amountsufficient to suppress, stabilize, reverse, slow or delay progression ofthe target infection or disease states. As such, the expression “aneffective amount” is used herein in reference to that quantity ofantagonist treatment which is necessary to obtain a reduction in thelevel of pathogen development and/or in the level of pathogenic diseaserelative to that occurring in an untreated control. Typically, aneffective amount of a given antagonist treatment provides a reduction ofat least 20%; or more typically, between 30 to 40%; more typically,between 50-60%; even more typically, between 70 to 80%; and even moretypically, between 90 to 95%, relative to the level of disease and/orthe level of pathogen development occurring in an untreated controlunder suitable conditions of treatment. An effective amount can beadministered in one or more administrations. The actual rate ofapplication of a liquid formulation will usually vary from a minimum ofabout 1×10³ to about 1×10¹⁰ viable cells/mL and preferably from about1×10⁶ to about 5×10⁹ viable cells/mL. Under most conditions, theantagonistic microbial strains of the invention described in theExamples below, would be optimally effective at application rates in therange of about 1×10⁶ to 1×10⁹ viable cells/mL, assuming a mode ofapplication which would achieve substantially uniform contact of atleast about 50% of the plant tissues. If the antagonists are applied asa solid formulation, the rate of application should be controlled toresult in a comparable number of viable cells per unit area of planttissue surface as obtained by the aforementioned rates of liquidtreatment. Typically, the biological control agents of the presentinvention are biologically effective when delivered at a concentrationin excess of 10⁶ CFU/g (colony forming units per gram), preferably inexcess of 10⁷ CFU/g, more preferably 10⁸ CFU/g, and most preferably at10⁹ CFU/g.

Further, the expression “effective microbial antagonist” used herein inreference to a microorganism is intended to mean that the subjectmicrobial strain exhibits a degree of inhibition of head blight diseaseexceeding, at a statistically significant level, that of an untreatedcontrol. Typically, an effective microbial antagonist has the ability toeffect a reduction of at least 20%; or more typically, between 30 to40%; more typically, between 50-60%; even more typically, between 70 to80%; and even more typically, between 90 to 95%, relative to the levelof disease and/or the level of pathogen development occurring in anuntreated control under suitable conditions of treatment.

Composition: A “composition” is intended to mean a combination of activeagent and at least another compound, carrier or composition, inert (forexample, a detectable agent or label or liquid carrier) or active, suchas a pesticide.

Isolated microbial strain, isolated culture, biologically pure culture,and enriched culture: As used herein, the term “isolated” as applied toa microorganism (e.g., bacterium or microfungus) refers to amicroorganism which has been removed and/or purified from an environmentin which it naturally occurs. As such, an “isolated strain” of a microbeas used herein is a strain that has been removed and/or purified fromits natural milieu. Thus, an “isolated microorganism” does not includeone residing in an environment in which it naturally occurs. Further,the term “isolated” does not necessarily reflect the extent to which themicrobe has been purified. A “substantially pure culture” of the strainof microbe refers to a culture which contains substantially no othermicrobes than the desired strain or strains of microbe. In other words,a substantially pure culture of a strain of microbe is substantiallyfree of other contaminants, which can include microbial contaminants aswell as undesirable chemical contaminants. Further, as used herein, a“biologically pure” strain is intended to mean the strain separated frommaterials with which it is normally associated in nature. Note that astrain associated with other strains, or with compounds or materialsthat it is not normally found with in nature, is still defined as“biologically pure.” A monoculture of a particular strain is, of course,“biologically pure.” As used herein, the term “enriched culture” of anisolated microbial strain refers to a microbial culture that containsmore than 50%, 60%, 70%, 80%, 90%, or 95% of the isolated strain.

As used herein, an “endophyte” is an endosymbiont that lives within aplant for at least part of its life without causing apparent disease.Endophytes may be transmitted either vertically (directly from parent tooffspring) or horizontally (from individual to unrelated individual).Vertically-transmitted fungal endophytes are typically asexual andtransmit from the maternal plant to offspring via fungal hyphaepenetrating the host's seeds. Bacterial endophytes can also betransferred vertically from seeds to seedlings (Ferreira et al., 2008).Conversely, horizontally-transmitted endophytes are typically sexual,and transmit via spores that can be spread by wind and/or insectvectors. Endophytes of crop plants have been receiving considerableattention with respect to their ability to control both disease andinsect infestation, as well as promoting plant growth.

Functionally comparable protein: The phrase “functionally comparableprotein” as used herein describes those proteins that have at least onecharacteristic in common. Such characteristics include sequencesimilarity, biochemical activity, transcriptional pattern similarity andphenotypic activity. Typically, the functionally comparable proteinsshare some sequence similarity or at least one biochemical. Within thisdefinition, homologs, orthologs, paralogs and analogs are considered tobe functionally comparable. In addition, functionally comparableproteins generally share at least one biochemical and/or phenotypicactivity. Functionally comparable proteins will give rise to the samecharacteristic to a similar, but not necessarily the same, degree.Typically, functionally comparable proteins give the samecharacteristics where the quantitative measurement due to one of thecomparables is at least 20% of the other; more typically, between 30 to40%; more typically, between 50-60%; even more typically, between 70 to80%; even more typically, between 90 to 95%; even more typically,between 98 to 100% of the other.

Fungicidal: As used herein, “fungicidal” refers to the ability of acomposition or substance to decrease the rate of growth of fungi or toincrease the mortality of fungi.

Fusarium fungus: For purposes of this invention it is understood thatthe use of term Fusarium fungus is intended to include both the sexual(teleomorphic) stage of this organism and also the asexual (anamorphic)stage, also referred to as the perfect and imperfect fungal stages,respectively. For example, the anamorphic stage of Fusarium graminearumis Gibberella zeae, a causative agent of head blight disease. Thisdisease typically occurs when the flower or seed head becomes inoculatedwith conidia produced by the imperfect form or ascospores produced bythe perfect form of this fungus.

Mutant: As used herein, the term “mutant” or “variant” in reference to amicroorganism refers to a modification of the parental strain in whichthe desired biological activity is similar to that expressed by theparental strain. For example, in the case of Microbacterium the“parental strain” is defined herein as the original Microbacteriumstrain before mutagenesis. Mutants or variants may occur in naturewithout the intervention of man. They also are obtainable by treatmentwith or by a variety of methods and compositions known to those of skillin the art. For example, a parental strain may be treated with achemical such as N-methyl-N′-nitro-N-nitrosoguanidine,ethylmethanesulfone, or by irradiation using gamma, X-ray, orUV-irradiation, or by other means well known to those practiced in theart.

Nematicidal: The term “nematicidal”, as used herein, refers to theability of a substance or composition to increase mortality or inhibitthe growth rate of nematodes.

Pathogen: The term “pathogen” as used herein refers to an organism suchas an alga, an arachnid, a bacterium, a fungus, an insect, a nematode, aparasitic plant, yeast, a protozoan, or a virus capable of producing adisease in a plant or animal. The term “phytopathogen” as used hereinrefers to a pathogenic organism that infects a plant.

Percentage of percent identity: “percentage of sequence identity”, asused herein, is determined by comparing two optimally locally alignedsequences over a comparison window defined by the length of the localalignment between the two sequences. The amino acid sequence in thecomparison window may comprise additions or deletions (e.g., gaps oroverhangs) as compared to the reference sequence (which does notcomprise additions or deletions) for optimal alignment of the twosequences. Local alignment between two sequences only includes segmentsof each sequence that are deemed to be sufficiently similar according toa criterion that depends on the algorithm used to perform the alignment(e.g. BLAST). The percentage identity is calculated by determining thenumber of positions at which the identical nucleic acid base or aminoacid residue occurs in both sequences to yield the number of matchedpositions, dividing the number of matched positions by the total numberof positions in the window of comparison and multiplying the result by100. Optimal alignment of sequences for comparison may be conducted bythe local homology algorithm of Smith and Waterman (1981) Add. APL.Math. 2:482, by the global homology alignment algorithm of Needleman andWunsch (J. Mal. Biol. 48:443, 1970), by the search for similarity methodof Pearson and Lipman (Proc. Natl. Acad. Sci. USA 85: 2444, 1988), byheuristic implementations of these algorithms (NCBI BLAST, WU-BLAST,BLAT, SIM, BLASTZ), or by inspection. Given that two sequences have beenidentified for comparison, GAP and BESTFIT are preferably employed todetermine their optimal alignment. Typically, the default values of 5.00for gap weight and 0.30 for gap weight length are used. The term“substantial sequence identity” between polynucleotide or polypeptidesequences refers to polynucleotide or polypeptide comprising a sequencethat has at least 50% sequence identity, preferably at least 70%,preferably at least 80%, more preferably at least 85%, more preferablyat least 90%, even more preferably at least 95%, and most preferably atleast 96%, 97%, 98% or 99% sequence identity compared to a referencesequence using the programs.

Query nucleic acid and amino acid sequences can be searched againstsubject nucleic acid or amino acid sequences residing in public orproprietary databases. Such searches can be done using the NationalCenter for Biotechnology Information Basic Local Alignment Search Tool(NCBI BLAST v 2.18) program. The NCBI BLAST program is available on theinternet from the National Center for Biotechnology Information(blast.ncbi.nlm.nih.gov/Blast.cgi). Typically the following parametersfor NCBI BLAST can be used: Filter options set to “default”, theComparison Matrix set to “BLOSUM62”, the Gap Costs set to “Existence:11, Extension: 1”, the Word Size set to 3, the Expect (E threshold) setto 1e-3, and the minimum length of the local alignment set to 50% of thequery sequence length. Sequence identity and similarity may also bedetermined using GenomeQuest™ software (Gene-IT, Worcester Mass. USA).

The term “pesticidal”, as used herein, refers to the ability of asubstance or composition to decrease the rate of growth of a pest, i.e.,an undesired organism, or to increase the mortality of a pest.

By “suppressive activity” of a biological control agent against aphytopathogen is meant the ability of the agent to suppress, inhibit,stabilize, reverse, slow, or delay the development of the pathogenitself, or the progression of the infection or disease states caused bythe pathogen.

Variant: A “variant”, as used herein in reference to a microorganism, isa strain having identifying characteristics of the species to which itbelongs, while having at least one nucleotide sequence variation oridentifiably different trait with respect to the parental strain, wherethe trait is genetically based (heritable). For example, for aMicrobacterium sp. SGI-014-006 strain having fungicidal activity,identifiable traits include 1) the ability to suppress the growth ofFusarium graminearum and its teleomorph Gibberella zeae; 2) the abilityto suppress the development of head blight disease; 3) havinghousekeeping genes with greater than 95%, greater than 96%, greater than97%, greater than 98%, or greater than 99% sequence identity to thehousekeeping genes of Microbacterium sp. SGI-014-006 can be used toconfirm a variant as Microbacterium sp. SGI-014-006.

For nucleic acids and polypeptides, the term “variant” is used herein todenote a polypeptide, protein or polynucleotide molecule with somedifferences, generated synthetically or naturally, in their amino acidor nucleic acid sequences as compared to a reference polypeptide orpolynucleotide, respectively. For example, these differences includesubstitutions, insertions, deletions or any desired combinations of suchchanges in a reference polypeptide or polypeptide. Polypeptide andprotein variants can further consist of changes in charge and/orpost-translational modifications (such as glycosylation, methylation,phosphorylation, etc.).

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

No admission is made that any reference constitutes prior art. Thediscussion of the references states what their authors assert, and theapplicants reserve the right to challenge the accuracy and pertinence ofthe cited documents. It will be clearly understood that, although anumber of prior art publications are referred to herein, this referencedoes not constitute an admission that any of these documents forms partof the common general knowledge in the art.

Methods for Taxonomic Identification

Microorganisms can often be distinguished based on direct microscopicanalysis (do all of the cells in a sample look the same on examination),staining characteristics, simple molecular analysis (such as a simplyrestriction fragment length polymorphism (RFLP) determination), and soforth. In addition to the illustrative examples of such taxonomicanalysis techniques as described at Examples 2-3 of the presentdisclosure, taxonomic identification of a microorganism can involve upto several different levels of analysis, and each analysis can be basedon a different characteristic of the organism. Such taxonomic analysescan include nucleic acid-based analysis (e.g., analysis of individualspecific genes, either as to their presence or their exact sequence, orexpression of a particular gene or a family of genes), protein-basedanalysis (e.g., at a functional level using direct or indirect enzymeassays, or at a structural level using immuno-detection techniques), andso forth.

a. Nucleic acid-based analysis: One of ordinary skill in the art willappreciate that a wide variety of nucleic acid-based techniques areknown and can be useful in obtaining the taxonomic identification of agiven microorganism. These techniques can be used to identify cells bygene sequence or to identify cells that have particular genes or genefamilies. Common gene families useful for taxonomic studies include 16Sgene family, actin gene family, and recombinase A (recA) gene family.These methods typically include amplifying and sequencing genes fromvery small numbers of cells, and therefore often overcome the problemsof concentrating cells and their DNA from dilute suspensions. The term“nucleic acid amplification” generally refers to techniques thatincrease the number of copies of a nucleic acid molecule in a sample orspecimen. Techniques useful for nucleic acid amplification are wellknown in the art. An example of nucleic acid amplification is thepolymerase chain reaction (PCR), in which a biological sample collectedfrom a subject is contacted with a pair of oligonucleotide primers,under conditions that allow for the hybridization of the primers tonucleic acid template in the sample. The primers are extended undersuitable conditions, dissociated from the template, and thenre-annealed, extended, and dissociated to amplify the number of copiesof the nucleic acid. Other examples of in vitro amplification techniquesinclude strand displacement amplification; transcription-free isothermalamplification; repair chain reaction amplification; ligase chainreaction; gap filling ligase chain reaction amplification; coupledligase detection and PCR; and RNA transcription-free amplification.

In addition to the illustrative example primers provided herein, see,e.g. Examples 2-3 and the Sequence Listing, primers have also beendesigned routinely, and new ones are continually being designed, forindividual species or phylogenetic groups of microorganisms. Suchnarrowly targeted primers can be used with the methods described hereinto screen and/or identify specifically only the microorganisms ofinterest.

Methods for preparing and using nucleic acid primers are described, forexample, in Sambrook et al. (In Molecular Cloning: A Laboratory Manual,CSHL, New York, 1989), Ausubel et al. (ed.) (In Current Protocols inMolecular Biology, John Wiley & Sons, New York, 1998). Amplificationprimer pairs can be derived from a known sequence, for example, by usingcomputer programs intended for that purpose such as Primer (WhiteheadInstitute for Biomedical Research, Cambridge, Mass.). One of ordinaryskill in the art will appreciate that the specificity of a particularprobe or primer increases with its length. Thus, for example, a primercomprising 30 consecutive nucleotides of an rRNA-encoding nucleotide orflanking region thereof will anneal to a target sequence with a higherspecificity than a corresponding primer of only 15 nucleotides. Thus, inorder to obtain greater specificity, probes and primers can be selectedthat comprise at least 20, 25, 30, 35, 40, 45, 50 or more consecutivenucleotides of a target nucleotide sequence such as the 16S rRNA.

Common techniques for the preparation of nucleic acids useful fornucleic acid applications (e.g., PCR) include phenol/chloroformextraction or use of one of the many DNA extraction kits that areavailable on the market. Another way that DNA can be amplified is byadding cells directly to the nucleic acid amplification reaction mix andrelying on the denaturation step of the amplification to lyse the cellsand release the DNA.

The product of nucleic acid amplification reactions may be furthercharacterized by one or more of the standard techniques that are wellknown in the art, including electrophoresis, restriction endonucleasecleavage patterns, oligonucleotide hybridization or ligation, and/ornucleic acid sequencing. When in hybridization techniques are used forcell identification purposes, a variety of probe labeling methods can beuseful, including fluorescent labeling, radioactive labeling andnon-radioactive labeling. When nucleic acid sequencing techniques areused, homology search for the nucleotide sequence of the amplifiednucleic acid molecules can be conducted using various databases of knownsequences, including but not limited to DDBJ/GenBank/EMBL databases.

b. Protein-based analysis: In addition to analysis of nucleic acids,microorganisms can be taxonomically characterized and identified basedon the presence (or absence) of specific proteins directly. Suchanalysis can be based on the activity of the specified protein, e.g.,through an enzyme assay or by the response of a co-cultured organisms,or by the mere presence of the specified protein (which can for instancebe determined using immunologic methods, such as in situimmunofluorescent antibody staining).

Enzyme assays: By way of example, fluorescent or chromogenic substrateanalogs can be included into the growth media (e.g., microtiter platecultures), followed by incubation and screening for reaction products,thereby identifying cultures on a basis of their enzymatic activities.

Co-cultivation response: In some embodiments of the present invention,the activity of an enzyme carried by a microbial isolate can be assayedbased on the response (or degree of response) of a co-cultured organism(such as a reporter organism).

A variety of methods can also be used for identifying microorganismsselected and isolated from a source environment by binding at least oneantibody or antibody-derived molecule to a molecule, or moreparticularly an epitope of a molecule, of the microorganism.

Anti-microorganism protein antibodies may be produced using standardprocedures described in a number of texts, including Harlow and Lane(Antibodies, A Laboratory Manual, CSHL, New York, 1988). Thedetermination that a particular agent binds substantially only to aprotein of the desired microorganism may readily be made by using oradapting routine procedures. One suitable in vitro assay makes use ofthe Western blotting procedure (described in many standard texts,including Harlow and Lane (Antibodies, A Laboratory Manual, CSHL, NewYork, 1988)).

Shorter fragments of antibodies (antibody-derived molecules, forinstance, FAbs, Fvs, and single-chain Fvs (SCFvs)) can also serve asspecific binding agents. Methods of making these fragments are routine.

Detection of antibodies that bind to cells on an array of this inventioncan be carried out using standard techniques, for instance ELISA assaysthat provide a detectable signal, for instance a fluorescent orluminescent signal.

Isolated Cultures of the Invention

As described in more details in the Examples section of the presentdisclosure, Applicants have discovered several agriculturally beneficialnovel microorganisms, for example, effective suppressors of head blightdisease. Particularly, these novel antagonistic microorganisms areeffective for reduction of head blight severity and for inhibition ofthe growth of Fusarium graminearum, a primary causative agent of headblight disease in wheat. The microbial antagonists were identified froma pool of approximately 5,000 microbial strains obtained from wild plantsamples collected from several locations in the United States. Initialselection of antagonistic microorganism was based on the ability of themicroorganisms to suppress the development of F. graminearum pathogenand that of its teleomorph Gibberella zeae in an in vitro antagonismassay. Selected microbial antagonists were then bio-assayed in agreenhouse study on wheat seedlings, which involved inoculation of theseedlings with the microbial strains, followed by repeated inoculationsof E graminearum spores, for the ability of the microbial strains toreduce the severity of fungal infestation and for their ability topreserve seed yield. The antagonistic microorganisms selected in thismanner were found to be effective in reducing the severity of headblight in greenhouse trials.

Taxonomic analysis further determined that each of the antagonisticmicroorganisms described in the present disclosure are closely relatedeither the bacterial genus Microbacterium, the bacterial genus Bacillus,the bacterial genus Variovorax, or the fungus genus Mycosphaerella.

The genus Microbacterium, the type genus of the familyMicrobacteriaceae, is generally considered to accommodate Gram-positive,non-spore forming, rod-shaped bacteria that were originally isolatedduring early studies on lactic acid-producing bacteria. Members of thegenus Microbacterium are originally characterized largely by theirmarked heat resistance, presence in dairies, and production of smallamounts of L(+) lactic acid from glucose. In contrast to other genera ofthe family Microbacteriaceae in which species are characterized by acoherent peptidoglycan type, species of Microbacterium possess ornithineor lysine either in the inter-peptide bridge or at position 3 of B-typepeptidoglycan. In other chemotaxonomic properties, such as isoprenoidquinones (MK-11, MK-12, MK-13), polar lipids, fatty acids and basecomposition of DNA, members of the genus exhibit the usual range ofdiversity found in other genera of Microbacteriaceae. Two bacterialgenera Microbacterium and Aureobacterium can be united according to sometaxonomy studies. To date, members of the genus Microbacterium compriseat least 33 species, which have been isolated from a broad range ofhabitats, including soil, dairy products, plant galls, insects orclinical specimens. Various aspects of their ecology, phylogeny,taxonomy, culture methods, and long-term preservation conditions haverecently been summarized by Evtushenko and Takeuchi (2006). One of skillin the art will readily appreciate that microorganism of the genusMicrobacterium can be taxonomically identified largely by any of thetaxonomic identification techniques described above, including thechemotaxonomic analysis of the cell wall peptidoglycan and comparative16S rDNA sequence analysis as described in Evtushenko and Takeuchi(2006) and references cited therein, as well as in those described inExamples 2-3 of the present disclosure. As discussed in detail below, todate several naturally occurring microorganisms have been reported ashaving antagonistic activity against head blight disease. However, thereare no reports prior to the present invention that describe amicroorganism of the genus Mycobacterium having such antagonisticactivity. Further, prior to the present invention, the present inventorswere not aware of any methods or processes of using a bacterial strainof the genus Mycobacterium as biocontrol agent in preventing, inhibitingor treating the development of a causative pathogen of head blightdisease.

The genus Bacillus is a genus of Gram-positive/variable, spore-forming,rod-shaped bacteria. Similarly to other genera associated with the earlyhistory of microbiology, such as Pseudomonas or Vibrio, nearly 266species members of the Bacillus genus are found ubiquitously, and it iswidely considered to be one of the genera with the largest 16S diversityand environmental diversity. Bacillus species can be obligate aerobes orfacultative anaerobes, and test positive for the enzyme catalase.Ubiquitous in nature, Bacillus includes both free-living and pathogenicspecies. Under stressful environmental conditions, the cells produceoval endospores that can stay dormant for extended periods. Thesecharacteristics originally defined the genus, but not all such speciesare closely related, and many have been moved to other genera. In fact,several studies have tried to reconstruct the phylogeny of the genus.The Bacillus-specific study with the most diversity covered is by Xu andCote [Intl. J. of Syst. Evol. Microbiol. 53 (3): 695-704; 2003], using16S and the ITS region, where they divide the genus into 10 groups,which includes the nested genera Paenibacillus, Brevibacillus,Geobacillus, Marinibacillus and Virgibacillus. However, according tomore recent studies, the genus Bacillus contains a very large number ofnested taxa and especially in both 16S and 23S it is considered to beparaphyletic to Lactobacillales (Lactobacillus, Streptococcus,Staphylococcus, Listeria, etc.), due to Bacillus coahuilensis and others(see, e.g., Yarda et al., Syst. Appl. Microbiol. 31 (4): 241-250, 2008;Yarda et al., Syst. Appl. Microbiol. 33 (6): 291-299, 2010]. Oneparticular Glade, formed by B. anthracis, B. cereus, B. mycoides, B.pseudomycoides, B. thuringiensis and B. weihenstephanensis under currentclassification standards, should be a single species (within 97% 16Sidentity), but due to medical reasons, they are considered separatespecies. In addition to the taxonomy analysis methods described atExamples 2-3 of the present disclosure, the phylogenetic and taxonomicanalyses of Bacillus species can be performed by a variety oftechniques, including those discussed in details in Xu and Cote, 2003;Yarda et al., 2008; Yarda et al., 2010.

The genus Variovorax was originally created by reclassification ofAlcaligenes paradoxus as Variovorax paradoxus (Willems et al., 1991),which is widely consider as the type species of this genus. V. paradoxushas been extensively studied as a model for novel biodegradation agents,as well as microbe/microbe and microbe/plant interactions. Other speciesinclude V. dokdonensis, V. soli (Kim et al., 2006), and V.boronicumulans (Miwa et al., 2008). Variovorax species are catabolicallyvery diverse and engage in mutually beneficial interactions with otherbacterial species in many biodegradations, and therefore possessecological importance and high application potential. For example, asoil methanotroph, only when co-cultured together with a V. paradoxusstrain, exhibits high affinity for methane (a potent greenhouse gas),and this trait is not usually observed in laboratory cultures.Similarly, a close relative of Variovorax has been found to be thecentral, non-photosynthetic partner within the phototrophic consortium“Chlorochromatium aggregatum.” Some species of the genus Variovorax alsohave the ability to interfere with the communication of other bacteria.Some yet other species of the genus Variovorax can intimately interactwith other biota (e.g., plants) in various ecosystems. Moreover, someVariovorax species, residing in the area just outside plant roots and/orinside a plant, have been reported for being capable of promoting plantgrowth via the reduction of ethylene levels, the repression ofquorum-sensing-controlled pathogenesis, and the increase of resistanceto heavy metals, which greatly benefits phytoremediation. In addition tothe taxonomy analysis methods described at Examples 2-3 of the presentdisclosure, the phylogenetic and taxonomic analyses of Variovoraxspecies can be performed by a variety of techniques, including thosediscussed in details elsewhere herein.

Mycosphaerella is a very large fungus genus, with over 2,000 speciesnames and at least 500 species associated with more than 40 anamorphgenera (especially Cercospora, Pseudocercospora, Septoria, Ramularia,etc.). In addition, several thousand anamorphs lack telomorphs. Thegenus of Mycosphaerella includes species that are pathogens, saprobes,endophytes, or mutualistic associations. Various aspects of theirecology, phylogeny, taxonomy, culture methods, and long-termpreservation conditions have recently been reported (see, e.g. Crous etal., Persoonia, 23:119-146,2009). One of skill in the art will readilyappreciate that microorganism of the genus Mycosphaerella can betaxonomically identified by any of the taxonomic techniques describedabove, or a combination thereof. Most common techniques includecomparative sequence analyses using 16S rDNA sequences and the internaltranscribed spacer regions as described in, for example, Crous et al.,Studies in Mycology, 55:235-253,2006; Crous et al., 2009, supra; Goodwinet al., Phytopathology 91: 648-658, 2001; and those described inExamples 2-3 of the present disclosure.

Deposit of Biological Material

Purified cultures of microbial strains identified as having suppressiveactivity against head blight disease were deposited in the AgriculturalResearch Service Culture Collection located at 1815 N. UniversityStreet, Peoria, Ill. 61604, USA (NRRL) in accordance with the BudapestTreaty for the purpose of patent procedure and the regulationsthereunder (Budapest Treaty). Accession numbers for these deposits areas follows:

SGI Strain ID Accession No Provisional Taxonomy SGI-005-G08 NRRL _-____Microbacterium sp. SGI-010-H11 NRRL 50471 Mycosphaerella sp. SGI-014-C06NRRL B-50470 Microbacterium sp. SGI-014-G01 NRRL B-50469 Variovorax sp.SGI-015-F03 NRRL B-50760 Bacillus sp. SGI-015-H06 NRRL B-50761 Bacillussp.

The microbial strains have been deposited under conditions that ensurethat access to the culture will be available during the pendency of thispatent application to one determined by the Commissioner of Patents andTrademarks to be entitled thereto under 37 C.F.R. § 1.14 and 35 U.S.C. §122. The deposits represent substantially pure cultures of the depositedstrains. The deposits are available as required by foreign patent lawsin countries wherein counterparts of the subject application or itsprogeny are filed. However, it should be understood that theavailability of a deposit does not constitute a license to practice thesubject invention in derogation of patent rights granted by governmentalaction.

Preferred microorganisms of the present invention have all of theidentifying characteristics of the deposited strains and, in particular,the identifying characteristics of being able to suppress thedevelopment of head blight disease as described herein, and as beingable to suppress the development of Fusarium graminearum pathogen andits teleomorph Gibberella zeae as described herein. In particular, thepreferred microorganisms of the present invention refer to the depositedmicroorganisms as described above, and mutants thereof

Microbiological Compositions

The microbiological compositions of the present invention that compriseisolated microbial strains or cultures thereof can be in a variety offorms, including, but not limited to, still cultures, whole cultures,stored stocks of cells, mycelium and/or hyphae (particularly glycerolstocks), agar strips, stored agar plugs in glycerol/water, freeze driedstocks, and dried stocks such as lyophilisate or mycelia dried ontofilter paper or grain seeds. As defined elsewhere herein, “isolatedculture” or grammatical equivalents as used in this disclosure and inthe art is understood to mean that the referred to culture is a culturefluid, pellet, scraping, dried sample, lyophilate, or section (forexample, hyphae or mycelia); or a support, container, or medium such asa plate, paper, filter, matrix, straw, pipette or pipette tip, fiber,needle, gel, swab, tube, vial, particle, etc. that contains a singletype of organism. In the present invention, an isolated culture of amicrobial antagonist is a culture fluid or a scraping, pellet, driedpreparation, lyophilate, or section of the microorganism, or a support,container, or medium that contains the microorganism, in the absence ofother organisms.

The present disclosure further provides compositions that contain atleast one isolated microbial strains or cultures thereof of the presentinvention and a carrier. The carrier may be any one or more of a numberof carriers that confer a variety of properties, such as increasedstability, wettability, dispersability, etc. Wetting agents such asnatural or synthetic surfactants, which can be nonionic or ionicsurfactants, or a combination thereof can be included in a compositionof the invention. Water-in-oil emulsions can also be used to formulate acomposition that includes at least one isolated microorganism of thepresent invention (see, for example, U.S. Pat. No. 7,485,451,incorporated by reference herein). Suitable formulations that may beprepared include wettable powders, granules, gels, agar strips orpellets, thickeners, and the like, microencapsulated particles, and thelike, liquids such as aqueous flowables, aqueous suspensions,water-in-oil emulsions, etc. The formulation may include grain or legumeproducts (e.g., ground grain or beans, broth or flour derived from grainor beans), starch, sugar, or oil. The carrier may be an agriculturalcarrier. In certain preferred embodiments, the carrier is a seed, andthe composition may be applied or coated onto the seed or allowed tosaturate the seed.

In some embodiments, the agricultural carrier may be soil or plantgrowth medium. Other agricultural carriers that may be used includewater, fertilizers, plant-based oils, humectants, or combinationsthereof. Alternatively, the agricultural carrier may be a solid, such asdiatomaceous earth, loam, silica, alginate, clay, bentonite,vermiculite, seed cases, other plant and animal products, orcombinations, including granules, pellets, or suspensions. Mixtures ofany of the aforementioned ingredients are also contemplated as carriers,such as but not limited to, pesta (flour and kaolin clay), agar orflour-based pellets in loam, sand, or clay, etc. Formulations mayinclude food sources for the cultured organisms, such as barley, rice,or other biological materials such as seed, plant parts, sugar canebagasse, hulls or stalks from grain processing, ground plant material(e.g. “yard waste”) or wood from building site refuse, sawdust or smallfibers from recycling of paper, fabric, or wood. Other suitableformulations will be known to those skilled in the art.

In the liquid form, e.g. solutions or suspensions, the microorganismsmay be mixed or suspended in water or in aqueous solutions. Suitableliquid diluents or carriers include water, aqueous solutions, petroleumdistillates, or other liquid carriers.

Solid compositions can be prepared by dispersing the antagonistmicroorganisms in and on an appropriately divided solid carrier, such aspeat, wheat, bran, vermiculite, clay, talc, bentonite, diatomaceousearth, fuller's earth, pasteurized soil, and the like. When suchformulations are used as wettable powders, biologically compatibledispersing agents such as non-ionic, anionic, amphoteric, or cationicdispersing and emulsifying agents can be used.

In a preferred embodiment, the compositions contemplated herein preventattack by head blight disease upon plants, particularly cereal plants,such as wheat, barley, oat, and corn and, when used in sufficientamounts, to act as microbial antagonists. These compositions, similarlyto other biocontrol agents, have a high margin of safety because theytypically do not burn or injury the plant.

As described in great detail throughout the present disclosure, controlof head blight disease may be effected by application of one or more ofthe microbiological compositions of the present invention to a hostplant or parts of the host plant. The compositions can be applied in anamount effective to reduce the level of head blight relative to that inan untreated control. The active constituents are used in aconcentration sufficient to inhibit plant pathogen development of thetargeted plant pathogen when applied to the cereal plant. As will beapparent to a skilled person in the art, effective concentrations mayvary depending upon such factors as: (a) the type of the plant oragricultural commodity; (b) the physiological condition of the plant oragricultural commodity; (c) the concentration of pathogens affecting theplant or agricultural commodity; (d) the type of disease injury on theplant or agricultural commodity; (e) weather conditions (e.g.temperature, humidity); and (f) the stage of plant disease. According tothe invention, typical concentrations are those higher than 1×10² CFU/mLof carrier. Preferred concentrations range from about 1×10⁴ to about1×10⁹ CFU/mL, such as the concentrations ranging from 1×10⁶ to 1×10⁸CFU/mL. More preferred concentrations are those of from about 35 toabout 150 mg dry microbial mass per gram of carrier (dry formulation) orper milliliter of carrier (liquid composition). In solid formulations,the rate of application should be controlled to result in a comparablenumber of viable cells per unit area of plant tissue surface as obtainedby the aforementioned rates of liquid treatment. Typically, thebiological control agents of the present invention are biologicallyeffective when delivered at a concentration in excess of 10⁶ CFU/g(colony forming units per gram), preferably in excess of 10⁷ CFU/g, morepreferably 10⁸ CFU/g, and most preferably at 10⁹ CFU/g.

In some embodiments, the amount of one or more of the biological controlagents in the microbial compositions of the present invention can varydepending on the final formulation as well as size or type of the plantor seed utilized. Preferably, the one or more biological control agentsin the microbial compositions are present in about 2% w/w/ to about 80%w/w of the entire formulation. More preferable, the one or morebiological control agents employed in the compositions is about 5% w/wto about 65% w/w and most preferably about 10% w/w to about 60% w/w byweight of the entire formulation.

As it will be appreciated by those skilled in the art, themicrobiological compositions of the invention may be applied to thewheat plant or other cereals using a variety of conventional methodssuch as dusting, coating, injecting, rubbing, rolling, dipping,spraying, or brushing, or any other appropriate technique which does notsignificantly injure the wheat plant or other cereals to be treated.Particularly preferred is the spray method.

Typically, the compositions of the invention are chemically inert; hencethey are compatible with substantially any other constituents of thespray schedule. They may also be used in combination with biologicallycompatible pesticidal active agents as for example, herbicides,nematicides, fungicides, insecticides, and the like. They can also beused in combination with plant growth affecting substances, such asfertilizers, plant growth regulators, and the like, provided that suchcompounds or substances are biologically compatible.

When used as pesticides or fungicide in their commercially availableformulations and in the use forms, prepared from these formulations, theactive microbial antagonists and compositions according to the presentinvention can furthermore be present in the form of a mixture withsynergists. Synergists are compounds by which the activity of the activecompositions is increased without it being necessary for the synergistadded to be active itself.

When used as pesticides in their commercially available formulations andin the use forms, prepared from these formulations, the active microbialantagonists and compositions according to the invention can furthermorebe present in the form of a mixture with inhibitors which reduce thedegradation of the active compositions after application in the habitatof the plant, on the surface of parts of plants or in plant tissues.

The active microbial antagonists and compositions according to theinvention, as such or in their formulations, can also be used as amixture with known acaricides, bactericides, fungicides, insecticides,microbicides, nematicides, pesticides, or combinations thereof, forexample in order to widen the spectrum of action or to prevent thedevelopment of resistances in this way. In many cases, synergisticeffects result, i.e. the activity of the mixture can exceed the activityof the individual components. A mixture with other known activecompounds, such as fertilizers, growth regulators, safeners and/orsemiochemicals is also contemplated.

In a preferred embodiment of the present invention, the compositions mayfurther include at least one chemical or biological pesticide. Theamount of at least one chemical or biological pesticide employed in thecompositions can vary depending on the final formulation as well as thesize of the plant and seed to be treated. Preferably, the at least onechemical or biological pesticide employed is about 0.1% w/w to about 80%w/w based on the entire formulation. More preferably, the at least onechemical or biological pesticide is present in an amount of about 1% w/wto about 60% w/w and most preferably about 10% w/w to about 50% w/w.

A variety of pesticides is apparent to one of skill in the art and maybe used. Exemplary chemical pesticides include those in the carbamate,organophosphate, organochlorine, and prethroid classes. Also includedare chemical control agents such as, but not limited to, benomyl, borax,captafol, captan, chorothalonil, formulations containing copper;formulations containing dichlone, dicloran, iodine, zinc; fungicidesthat inhibit ergosterol biosynthesis such as but not limited toblastididin, cymoxanil, fenarimol, flusilazole, folpet, imazalil,ipordione, maneb, manocozeb, metalaxyl, oxycarboxin, myclobutanil,oxytetracycline, PCNB, pentachlorophenol, prochloraz, propiconazole,quinomethionate, sodium aresenite, sodium DNOC, sodium hypochlorite,sodium phenylphenate, streptomycin, sulfur, tebuconazole, terbutrazole,thiabendazolel, thiophanate-methyl, triadimefon, tricyclazole,triforine, validimycin, vinclozolin, zineb, and ziram. A variety ofinsecticidal compounds can be useful for the compositions of theinvention, including but not limited to those cited in U.S. Pat. Appl.No. 20110033432A1.

The microbiological compositions of the present invention preferablyinclude at least one biological pesticide. Exemplary biologicalpesticides that are suitable for use herein and can be included in amicrobiological composition according to the present invention forpreventing a plant pathogenic disease include microbes, animals,bacteria, fungi, genetic material, plant, and natural products of livingorganisms. In these compositions, the microorganism of the presentinvention is isolated prior to formulation with an additional organism.For example, microbes such as but not limited to species of Ampelomyces,Aureobasidium, Bacillus, Beauveria, Candida, Chaetomium, Cordyceps,Cryptococcus, Dabaryomyces, Erwinia, Exophilia, Gliocladium, Mariannaea,Paecilomyces, Paenibacillus, Pantoea, Pichia, Pseudomonas,Sporobolomyces, Talaromyces, and Trichoderma can be provided in acomposition with the antagonistic microorganisms of the presentinvention, with fungal strains of the Muscodor genus being particularlypreferred. Use of the microbiological compositions according to thepresent invention in combination with the microbial antagonistsdisclosed in U.S. Pat. Nos. 7,518,040; 7,601,346; 6,312,940 is alsoparticularly preferred.

Examples of fungi that can be combined with microbial antagonists andcompositions of the present invention in a composition include, withoutlimitation, Muscodor species, Aschersonia aleyrodis, Beauveria bassiana(“white muscarine”), Beauveria brongniartii, Chladosporium herbarum,Cordyceps clavulata, Cordyceps entomorrhiza, Cordyceps facis, Cordycepsgracilis, Cordyceps melolanthae, Cordyceps militaris, Cordycepsmyrmecophila, Cordyceps ravenelii, Cordyceps sinensis, Cordycepssphecocephala, Cordyceps subsessilis, Cordyceps unilateralis, Cordycepsvariabilis, Cordyceps washingtonensis, Culicinomyces clavosporus,Entomophaga grylli, Entomophaga maimaiga, Entomophaga muscae,Entomophaga praxibulli, Entomophthora plutellae, Fusarium lateritium,Hirsutella citriformis, Hirsutella thompsoni, Metarhizium anisopliae(“green muscarine”), Metarhizium flaviride, Muscodor albus,Neozygitesfloridana, Nomuraea rileyi, Paecilomyces farinosus,Paecilomyces fumosoroseus, Pandora neoaphidis, Tolypocladiumcylindrosporum, Verticillium lecanii, Zoophthora radicans, andmycorrhizal species such as Laccaria bicolor. Other mycopesticidalspecies will be apparent to those skilled in the art.

The present invention also provides methods of treating a plant byapplication of any of a variety of customary formulations in aneffective amount to either the soil (i.e., in-furrow), a portion of theplant (i.e., drench) or on the seed before planting (i.e., seed coatingor dressing). Customary formulations include solutions, emulsifiableconcentrate, wettable powders, suspension concentrate, soluble powders,granules, suspension-emulsion concentrate, natural and syntheticmaterials impregnated with active compound, and very fine controlrelease capsules in polymeric substances. In certain embodiments of thepresent invention, the biological control compositions are formulated inpowders that are available in either a ready-to-use formulation or aremixed together at the time of use. In either embodiment, the powder maybe admixed with the soil prior to or at the time of planting. In analternative embodiment, one or both of either the biological controlagent or insect control agent is a liquid formulation that is mixedtogether at the time of treating. One of ordinary skill in the artunderstands that an effective amount of the inventive compositionsdepends on the final formulation of the composition as well as the sizeof the plant or the size of the seed to be treated.

Depending on the final formulation and method of application, one ormore suitable additives can also be introduced to the compositions ofthe present invention. Adhesives such as carboxymethylcellulose andnatural and synthetic polymers in the form of powders, granules orlatexes, such as gum arabic, chitin, polyvinyl alcohol and polyvinylacetate, as well as natural phospholipids, such as cephalins andlecithins, and synthetic phospholipids, can be added to the presentcompositions.

In a preferred embodiment, the microbiological compositions areformulated in a single, stable solution, or emulsion, or suspension. Forsolutions, the active chemical compounds (i.e., the pest control agents)are typically dissolved in solvents before the biological control agentis added. Suitable liquid solvents include petroleum based aromatics,such as xylene, toluene or alkylnaphthalenes, aliphatic hydrocarbons,such as cyclohexane or paraffins, for example petroleum fractions,mineral and vegetable oils, alcohols, such as butanol or glycol as wellas their ethers and esters, ketones, such as methyl ethyl ketone, methylisobutyl ketone or cyclohexanone, strongly polar solvents, such asdimethylformamide and dimethyl sulphoxide. For emulsion or suspension,the liquid medium is water. In one embodiment, the chemical controlagent and biological control agent are suspended in separate liquids andmixed at the time of application. In a preferred embodiment ofsuspension, the insect control agent and biologic are combined in aready-to-use formulation that exhibits a shelf-life of at least twoyears. In use, the liquid can be sprayed or can be applied foliarly asan atomized spray or in-furrow at the time of planting the crop. Theliquid composition can be introduced to the soil before germination ofthe seed or directly to the soil in contact with the roots by utilizinga variety of techniques known in the art including, but not limited to,drip irrigation, sprinklers, soil injection or soil drenching.

Optionally, stabilizers and buffers can be added, including alkaline andalkaline earth metal salts and organic acids, such as citric acid andascorbic acid, inorganic acids, such as hydrochloric acid or sulfuricacid. Biocides can also be added and can include formaldehydes orformaldehyde-releasing agents and derivatives of benzoic acid, such asp-hydroxybenzoic acid.

Seed Coating Formulations

In some particularly preferred embodiments, the biocontrol compositionsof the present invention are formulated as a seed treatment. The seedtreatment preferably comprises at least one insect control agent and atleast one biological control agent. It is contemplated that the seedscan be substantially uniformly coated with one or more layers of thebiocontrol compositions disclosed herein using conventional methods ofmixing, spraying or a combination thereof through the use of treatmentapplication equipment that is specifically designed and manufactured toaccurately, safely, and efficiently apply seed treatment products toseeds. Such equipment uses various types of coating technology such asrotary coaters, drum coaters, fluidized bed techniques, spouted beds,rotary mists or a combination thereof. Liquid seed treatments such asthose of the present invention can be applied via either a spinning“atomizer” disk or a spray nozzle which evenly distributes the seedtreatment onto the seed as it moves though the spray pattern.Preferably, the seed is then mixed or tumbled for an additional periodof time to achieve additional treatment distribution and drying. Theseeds can be primed or unprimed before coating with the inventivecompositions to increase the uniformity of germination and emergence. Inan alternative embodiment, a dry powder formulation can be metered ontothe moving seed and allowed to mix until completely distributed.

Another aspect of the invention provides seeds treated with the subjectmicrobiological compositions. One embodiment provides seeds having atleast part of the surface area coated with a microbiological compositionaccording to the present invention. In a specific embodiment, themicroorganism-treated seeds have a spore concentration or microbial cellconcentration from about 10⁶ to about 10⁹ per seed. The seeds may alsohave more spores or microbial cells per seed, such as, for example 10¹⁰,10¹¹ or 10¹² spores per seed. The microbial spores and/or cells can becoated freely onto the seeds or, preferably, they can be formulated in aliquid or solid composition before being coated onto the seeds. Forexample, a solid composition comprising the microorganisms can beprepared by mixing a solid carrier with a suspension of the spores untilthe solid carriers are impregnated with the spore or cell suspension.This mixture can then be dried to obtain the desired particles.

In some other embodiments, it is contemplated that the solid or liquidbiocontrol compositions of the present invention further containfunctional agents capable of protecting seeds from the harmful effectsof selective herbicides such as activated carbon, nutrients(fertilizers), and other agents capable of improving the germination andquality of the products or a combination thereof.

Seed coating methods and compositions that are known in the art can beparticularly useful when they are modified by the addition of one of theembodiments of the present invention. Such coating methods and apparatusfor their application are disclosed in, for example, U.S. Pat. Nos.5,918,413; 5,554,445; 5,389,399; 4,759,945; and 4,465,017. Various seedcoating compositions are disclosed, for example, in U.S. Pat. Appl. Nos.US20110033432, US20100154299, U.S. Pat. Nos. 5,939,356; 5,876,739,5,849,320; 5,791,084, 5,661,103; 5,580,544, 5,328,942; 4,735,015;4,634,587; 4,372,080, 4,339,456; and 4,245,432, among others.

A variety of additives can be added to the seed treatment formulationscomprising the inventive compositions. Binders can be added and includethose composed preferably of an adhesive polymer that can be natural orsynthetic without phytotoxic effect on the seed to be coated. The bindermay be selected from polyvinyl acetates; polyvinyl acetate copolymers;ethylene vinyl acetate (EVA) copolymers; polyvinyl alcohols; polyvinylalcohol copolymers; celluloses, including ethylcelluloses,methylcelluloses, hydroxymethylcelluloses, hydroxypropylcelluloses andcarboxymethylcellulose; polyvinylpyrolidones; polysaccharides, includingstarch, modified starch, dextrins, maltodextrins, alginate andchitosans; fats; oils; proteins, including gelatin and zeins; gumarabics; shellacs; vinylidene chloride and vinylidene chloridecopolymers; calcium lignosulfonates; acrylic copolymers;polyvinylacrylates; polyethylene oxide; acrylamide polymers andcopolymers; polyhydroxyethyl acrylate, methylacrylamide monomers; andpolychloroprene.

Any of a variety of colorants may be employed, including organicchromophores classified as nitroso; nitro; azo, including monoazo,bisazo and polyazo; acridine, anthraquinone, azine, diphenylmethane,indamine, indophenol, methine, oxazine, phthalocyanine, thiazine,thiazole, triarylmethane, xanthene. Other additives that can be addedinclude trace nutrients such as salts of iron, manganese, boron, copper,cobalt, molybdenum and zinc. A polymer or other dust control agent canbe applied to retain the treatment on the seed surface.

In some specific embodiments, in addition to the microbial cells orspores, the coating can further comprise a layer of adherent. Theadherent should be non-toxic, biodegradable, and adhesive. Examples ofsuch materials include, but are not limited to, polyvinyl acetates;polyvinyl acetate copolymers; polyvinyl alcohols; polyvinyl alcoholcopolymers; celluloses, such as methyl celluloses, hydroxymethylcelluloses, and hydroxymethyl propyl celluloses; dextrins; alginates;sugars; molasses; polyvinyl pyrrolidones; polysaccharides; proteins;fats; oils; gum arabics; gelatins; syrups; and starches. More examplescan be found in, for example, U.S. Pat. No. 7,213,367 and U.S. Pat.Appln. No. US20100189693.

Various additives, such as adherents, dispersants, surfactants, andnutrient and buffer ingredients, can also be included in the seedtreatment formulation. Other conventional seed treatment additivesinclude, but are not limited to, coating agents, wetting agents,buffering agents, and polysaccharides. At least one agriculturallyacceptable carrier can be added to the seed treatment formulation suchas water, solids or dry powders. The dry powders can be derived from avariety of materials such as calcium carbonate, gypsum, vermiculite,talc, humus, activated charcoal, and various phosphorous compounds.

In some embodiment, the seed coating composition can comprise at leastone filler which is an organic or inorganic, natural or syntheticcomponent with which the active components are combined to facilitateits application onto the seed. Preferably, the filler is an inert solidsuch as clays, natural or synthetic silicates, silica, resins, waxes,solid fertilizers (for example ammonium salts), natural soil minerals,such as kaolins, clays, talc, lime, quartz, attapulgite,montmorillonite, bentonite or diatomaceous earths, or syntheticminerals, such as silica, alumina or silicates, in particular aluminumor magnesium silicates.

The seed treatment formulation may further include one or more of thefollowing ingredients: other pesticides, including compounds that actonly below the ground; fungicides, such as captan, thiram, metalaxyl,fludioxonil, oxadixyl, and isomers of each of those materials, and thelike; herbicides, including compounds selected from glyphosate,carbamates, thiocarbamates, acetamides, triazines, dinitroanilines,glycerol ethers, pyridazinones, uracils, phenoxys, ureas, and benzoicacids; herbicidal safeners such as benzoxazine, benzhydryl derivatives,N,N-diallyl dichloroacetamide, various dihaloacyl, oxazolidinyl andthiazolidinyl compounds, ethanone, naphthalic anhydride compounds, andoxime derivatives; fertilizers; and biocontrol agents such as othernaturally-occurring or recombinant bacteria and fungi from the generaRhizobium, Bacillus, Pseudomonas, Serratia, Trichoderma, Glomus,Gliocladium and mycorrhizal fungi. These ingredients may be added as aseparate layer on the seed or alternatively may be added as part of theseed coating composition of the invention.

Preferably, the amount of the novel composition or other ingredientsused in the seed treatment should not inhibit germination of the seed,or cause phytotoxic damage to the seed.

The microorganism-treated seeds may also be further enveloped with afilm overcoating to protect the coating. Such overcoatings are known inthe art and may be applied using fluidized bed and drum film coatingtechniques.

In principle, any plant seed capable of germinating to form a plant thatis susceptible to attack by nematodes and/or pathogenic fungi can betreated in accordance with the invention. Suitable seeds include thoseof cereals, coffee, cole crops, fiber crops, flowers, fruits, legume,oil crops, trees, tuber crops, vegetables, as well as other plants ofthe monocotyledonous, and dicotyledonous species. Preferably, crop seedsare coated which include but are not limited to bean, carrot, corn,cotton, grasses, lettuce, peanut, pepper, potato, rapeseed, rice, rye,sorghum, soybean, sugar beet, sunflower, tobacco, and tomato seeds. Mostpreferably, barley or wheat (spring wheat or winter wheat) seeds arecoated with the present compositions.

Also provided, in another aspect of the present invention, is a novelcereal plant created by artificially introducing a microbial endophyteof the invention into a cereal plant that is free of endophyticmicroorganisms. In some embodiments of this aspect, the microbialendophyte introduced into the cereal plant may be an endophyticantagonist having suppressive activity against head blight disease or acausative agent of head blight disease. Furthermore, the endophyticantagonist introduced into the cereal plant may be the fungal strainSGI-010-H11. A variety of methods previously found effective for theintroduction of a microbial endophyte into cereal grass species areknown in the art. Examples of such methods include those described inU.S. Pat. Appl. No. 20030195117A1, U.S. Pat. Appl. No. 20010032343A1,and U.S. Pat. No. 7,084,331, among others. It will become apparent tothose skilled in the art that many of the aforementioned methods can beuseful for the making of a novel cereal plant of the invention.

After artificial infection, it is preferred that DNA of the isolatedendophytic antagonist is amplified by PCR and the antagonist isconfirmed by carrying out a homology search for the DNA amplified.Further it is preferred that a foreign gene that expresses anidentifiable means is introduced into the above-mentioned endophyticantagonist, and the presence of the colonization of the above-mentionedendophytic antagonist infecting the plant is confirmed by theabove-identifiable means using the foreign gene.

Preparing the Biocontrol Compositions According to the Present Invention

Cultures of the microbial antagonists may be prepared for use in thebiocontrol compositions of the invention using standard static dryingand liquid fermentation techniques known in the art. Growth is commonlyeffected in a bioreactor.

A bioreactor refers to any device or system that supports a biologicallyactive environment. As described herein a bioreactor is a vessel inwhich microorganisms including the microbial antagonists of theinvention can be grown. A bioreactor may be any appropriate shape orsize for growing the microorganisms. A bioreactor may range in size andscale from 10 mL to liter's to cubic meters and may be made of stainlesssteel or any other appropriate material as known and used in the art.The bioreactor may be a batch type bioreactor, a fed batch type or acontinuous-type bioreactor (e.g., a continuous stirred reactor). Forexample, a bioreactor may be a chemostat as known and used in the art ofmicrobiology for growing and harvesting bacteria. A bioreactor may beobtained from any commercial supplier (See also Bioreactor SystemDesign, Asenjo & Merchuk, CRC Press, 1995).

For small scale operations, a batch bioreactor may be used, for example,to test and develop new processes, and for processes that cannot beconverted to continuous operations.

Microorganisms grown in a bioreactor may be suspended or immobilized.Growth in the bioreactor is generally under aerobic conditions atsuitable temperatures and pH for growth. For the organisms of theinvention, cell growth can be achieved at temperatures between 5 and 37°C., with the preferred temperature being in the range of 15 to 30° C.,15 to 28° C., 20 to 30° C., or 15 to 25° C. The pH of the nutrientmedium can vary between 4.0 and 9.0, but the preferred operating rangeis usually slightly acidic to neutral at pH 4.0 to 7.0, or 4.5 to 6.5,or pH 5.0 to 6.0. Typically, maximal cell yield is obtained in 20-72hours after inoculation.

Optimal conditions for the cultivation of antagonists of this inventionwill, of course, depend upon the particular strain. However, by virtueof the conditions applied in the selection process and generalrequirements of most microorganisms, a person of ordinary skill in theart would be able to determine essential nutrients and conditions. Themicrobial antagonists would typically be grown in aerobic liquidcultures on media which contain sources of carbon, nitrogen, andinorganic salts that can be assimilated by the microorganism andsupportive of efficient cell growth. Preferred carbon sources arehexoses such as glucose, but other sources that are readily assimilatedsuch as amino acids, may be substituted. Many inorganic andproteinaceous materials may be used as nitrogen sources in the growthprocess. Preferred nitrogen sources are amino acids and urea but othersinclude gaseous ammonia, inorganic salts of nitrate and ammonium,vitamins, purines, pyrimidines, yeast extract, beef extract, proteosepeptone, soybean meal, hydrolysates of casein, distiller's solubles, andthe like. Among the inorganic minerals that can be incorporated into thenutrient medium are the customary salts capable of yielding calcium,zinc, iron, manganese, magnesium, copper, cobalt, potassium, sodium,molybdate, phosphate, sulfate, chloride, borate, and like ions. Withoutbeing limited thereto, use of potato dextrose liquid medium for fungalantagonists and R2A broth premix for bacterial strains is preferred.

Throughout this disclosure, various information sources are referred toand incorporated by reference. The information sources include, forexample, scientific journal articles, patent documents, textbooks, andWorld Wide Web browser-inactive page addresses. The reference to suchinformation sources is solely for the purpose of providing an indicationof the general state of the art at the time of filing. While thecontents and teachings of each and every one of the information sourcescan be relied on and used by one of skill in the art to make and useembodiments of the invention, any discussion and comment in a specificinformation source should in no way be considered as an admission thatsuch comment was widely accepted as the general opinion in the field.

The discussion of the general methods given herein is intended forillustrative purposes only. Other alternative methods and embodimentswill be apparent to those of skill in the art upon review of thisdisclosure, and are to be included within the spirit and purview of thisapplication.

It should also be understood that the following examples are offered toillustrate, but not limit, the invention.

EXAMPLES Example 1: Discovery of the Antagonistic Microorganisms Capableof Suppressing the Development of Fusarium graminaerum and Gibberellazeae, Causative Agents of Head Blight Disease

This example describes a high-throughput process of collecting andscreening candidate microorganisms that has been developed internally atSynthetic Genomics, Inc. to isolate strains of microorganisms havingsuppressive activity against causative agents of head blight disease,particularly against Fusarium graminearum. Novel microbial antagonisticstrains were isolated from plant tissue samples collected from severallocations across the United States. The bacterial strains SGI-014-006,SGI-014-G01, SGI-015-F03, and SGI-015-H06 were isolated from plant roottissues collected from Eagle Peak Preserve near Julian, Calif. Thefungal strain SGI-010-H11 and the bacterial strain SGI-005-G08 wereisolated from stem tissues of two different plant samples collected fromSan Elijo Lagoon, Encinitas, Calif.

The microorganisms were isolated as follows. For bacterial strainisolation, plant root tissues were sonicated and subjected to serialdilutions on 2×YT (yeast extract and tryptone) and N-free agar mediumplates. Individual colonies were then selected based on morphologicalcharacteristics, and individually cultured in liquid broth medium. Forfungal isolation, plant tissue was first surface-sterilized by dippingin 70% ethanol and briefly passing through a flame. The tissue was thendissected and placed on potato dextrose agar (PDA) medium, followed byincubation at room temperature. When mycelial growth was observed, asegment of mycelial growth was then transferred to another PDA plate.Strains of microorganisms were isolated, purified and preserved at −80°C. in 15% glycerol at for bacterial strains and on dried barley seed forfungal strains.

Strains of microorganisms isolated as described above were thereafterassayed for their ability to suppress mycelial growth of F. graminearumin an in vitro antagonism test, which was performed on agar platescontaining potato dextrose agar (PDA) growth medium according to ahigh-throughput screening procedure described in U.S. Pat. Appl. No.US20120107915A1, which is incorporated by reference herein, with minormodifications. Briefly, isolated strains of microorganisms were grown onone-fifth strength Tryptic soy broth agar (TSBA/5) for 24 hours prior touse. Conidial inoculum of E graminearum NRRL-5883 was produced by hyphaltipping an actively growing colony of the fungus and transferring thehyphal strands to PDA agar medium. After incubating the plates for 7days at 25° C. using a 12 h/day photoperiod, conidia were washed fromPDA plates using a weak phosphate buffer (0.004% phosphate buffer, pH7.2, with 0.019% MgCl₂). A suspension of conidia of E graminearum in theweak phosphate buffer (1×10⁵ conidia/ml) was then immediately spreadover the agar surface, and the plates were then incubated at 25° C. for48-72 hours. To initiate the antagonism test, cells of isolatedmicrobial strains were point-inoculated at equal distances inside theperimeter of the plate. After five days, the strains were scored asantibiosis positive when a visibly clear area that lacked mycelialgrowth existed around the perimeter of the microbial colonies.

More than 4,000 microbial strains were isolated and assayed by theprocedure described above. Of those, six strains were found tosignificantly suppress the mycelial growth of F. graminearum NRRL-5883on PDA medium. These new microbial antagonists are identified asSGI-005-G08, SGI-010-H11, SGI-014-006, SGI-014-G01, SGI-015-F03, andSGI-015-H06.

Antagonism test was also performed for the new microbial antagonists fortheir ability to suppress the growth of fungal pathogen Gibberella zeae,which is a teleomorphic species of Fusarium graminearum. All procedureswere identical to those described above, except that the pathogen straintested in this assay was a pathogenic strain of the fungus Gibberellazeae. Of these six microbial antagonists, four strains SGI-010-H11,SGI-014-006, SGI-015-F03, and SGI-015-1106 were found to significantlysuppress the mycelial growth of Gibberella zeae.

Example 2: DNA Extraction, Sequencing and Taxonom

Fungal Cell Lysis and Acquiring ITS-5.8S rDNA Sequence Information

The fungal biomass was transferred to a 96-well PCR microplatecontaining 50 μl of a 2× lysis buffer (100 mM Tris HCL, pH 8.0, 2 mMEDTA, pH 8.0, 1% SDS, 400 μg/ml Proteinase K). Lysis conditions were asfollows: 55° C. for 60 minutes, 94° C. for 4 minutes. An aliquot of thelysis product was used as the source of template DNA for PCRamplification. The ITS-5.8S rDNA sequence was amplified via PCR usingtwo primers M13-ITS1 (SEQ ID NO: 15) and ITS4 M13-tailed (SEQ ID NO:16).

For amplification of the ITS-5.8S rDNA region, each PCR mixture wasprepared in a 20-μ1 final volume reaction containing 4 μl from thefungal lysis reaction, 0.2 μM of each primer (ITS1/ITS4), 6% Tween-20,and 10 μl of 2× ImmoMix (Bioline USA Inc, Taunton, Mass.). PCRconditions were as follows: 94° C. for 10 minutes; 94° C. for 30seconds, 52° C. for 30 seconds, 72° C. for 75 seconds for 30 cycles; 72°C. for 10 minutes. A 2-μl aliquot of the PCR product was run on a 1.0%agarose gel to confirm a single band of the expected size. Positivebands were sent out for Sanger sequencing in the forward and reversedirections using M13 primers. Sequence of the 5.8S intergenic region(ITS) of the fungal strain SGI-010-H11 is provided in the SequenceListing as SEQ ID NO: 11. Homology search for the determined nucleotidesequence of the 5.8S intergenic region (ITS) was conducted using theDDBJ/GenBank/EMBL database. Subsequently, the phylogenetic relationshipof the nucleotide sequence of the 5.8S intergenic region (ITS) wasanalyzed among the isolated fungal antagonist SGI-010-H11 describedherein, microorganisms of the genera and species that exhibit highsequence homologies to the isolated fungal antagonist SGI-010-H11, andother wide varieties of microorganism genera and species, using theClustalW phylogenetic tree building program. The fungal strainSGI-010-H11 is considered to be related to the family ofMycosphaerellaceae based upon ˜97% similarity of its ITS-5.8S rDNAsequence to those of Mycosphaerella punctiformis (GenBank EU343182) andRamularia pratensis (GenBank EU019284), whose 5.8S ITS sequences showclear relatedness to Mycosphaerellaceae.

Bacterial Cell Lysis and Acquiring 16S rRNA Sequence Information

A 20 μl aliquot of cell suspension was transferred to a 96-well PCRplate containing 20 μl of a 2× lysis buffer (100 mM Tris HCL, pH 8.0, 2mM EDTA, pH 8.0, 1% SDS, 400 μg/ml Proteinase K). Lysis conditions wereas follows: 55° C. for 30 minutes, 94° C. for 4 minutes. An aliquot ofthe lysis product was used as the source of template DNA for PCRamplification. The 16S rRNA sequence was amplified via PCR using M13-27F(SEQ ID NO: 17) and 1492R M13-tailed (SEQ ID NO: 18) primers.

For amplification of 16S rRNA region, each PCR mixture was prepared in a20-μl final volume reaction containing 4 μl from the bacterial lysisreaction, 2 μM of each primer (27F/1492R), 6% Tween-20, and 10 μl of 2×ImmoMix (Bioline USA Inc, Taunton, Mass.). PCR conditions were asfollows: 94° C. for 10 minutes; 94° C. for 30 seconds, 52° C. for 30seconds, 72° C. for 75 seconds for 30 cycles; 72° C. for 10 minutes. A2-μl aliquot of the PCR product was run on a 1.0% agarose gel to confirma single band of the expected size. Positive bands were sent out forSanger sequencing in the forward and reverse directions using M13primers. Sequences of the 16S rRNA of the bacterial strains SGI-014-006,SGI-005-G08, SGI-014-G01, SGI-015-F03, and SGI-015-H06 are approximately1.4-Kb in length and are provided in the Sequence Listing as SEQ ID NOs:1, 10, 12, 13, and 14 respectively. Homology search for the determinednucleotide sequence was conducted using the DDBJ/GenBank/EMBL database.Subsequently, the phylogenetic relationship of the nucleotide sequenceof the 16 rRNA genes was analyzed among the isolated bacterialantagonists described herein, bacteria of the genera and species thatexhibit high sequence homologies to the isolated bacterial antagonists,and other wide varieties of bacterial genera and species, using theClustalW phylogenetic tree building program. Sequence identity andsimilarity were also determined using GenomeQuest™ software (Gene-IT,Worcester Mass. USA). The sequence analysis result revealed that thebacterial isolate SGI-014-G01 can be considered to be related to thegenus of Variovorax based upon ˜99% similarity of its 16S rRNA sequenceto those of several Variovorax species, including Variovorax sp. R-21938(GenBank AJ786799) and Variovorax paradoxus (GenBank GU186109), whose16S rRNA sequences show clear relatedness to Variovorax sp. Further, thesequence analysis result also revealed that the two bacterial isolatesSGI-015-F03 and SGI-015-H06 can be considered to be related to thefamily of Bacillaceae, based upon >99% similarity of their respective16S sequences to those of several Bacillus spp.

The sequence analysis result revealed that the two bacterial isolatesSGI-014-006 and SGI-055-G08 can be considered to be related to thefamily of Microbacteriaceae based upon >99% similarity of theirrespective 16S sequences to those of several Microbacterium spp.Notably, the 1430-nt sequence of the 16S rRNA gene of SGI-014-006 (SEQID NO: 1) is identical to the 16S rRNA gene of a Microbacterium oxydansstrain DSM 20578 and several other Microbacterium sp. strains (e.g.,GenBank sequences Y17227.1, FJ200406, EU086800, and EU714335) over itsentire 1430-nt length.

In particular, among the thousands of microbial strains that wereisolated and tested for the ability to suppress Fusarium development inantagonism assays as described in Example 1, a total of eighty-eightstrains was subsequently identified as Microbacterium species based uponthe sequence similarity of their 16S sequences to those of knownMicrobacterium spp. However, Applicants have found that SGI-014-006 andSGI-055-G08 were the only two Microbacterium strains that possessedsuppressive activity against Fusarium graminearum. To date, as discussedabove, several naturally occurring microbes have been reported as havingantagonistic activity against head blight disease. However, there are noreports prior to the present invention that describe a microorganism ofthe genus Mycobacterium having such antagonistic activity. Further,prior to the present invention, the present inventors were not aware ofany methods or processes of using a bacterial strain of the genusMycobacterium as biocontrol agent in preventing, inhibiting or treatingthe development of a causative pathogen of head blight disease.

Example 3: Sequence Analysis of Housekeeping Genes from the IsolateSGI-014-C06

A phylogenetic study of several housekeeping genes from 27 species ofthe genus Microbacterium has been reported recently by Richert et al.(Syst. Appl. Microbiol. 30:102-108, 2007). The study concluded thatalthough the merits of the 16S rRNA sequence analysis for systematictaxonomy are unsurpassed, sole emphasis on a single taxonomic parametershould not guide systematic conclusions. As disclosed above, thenucleotide sequence of the 16S rRNA gene of SGI-014-006 (SEQ ID NO: 1)is identical to the 16S rRNA gene of several Microbacterium sp. strains,including the nucleotide sequence of a 16S rRNA gene of a Microbacteriumoxydans strain DSM 20578 that was included in Richert et al. (2007)study (GenBank Accession Y17227.1). Applicants proceeded to perform aphylogenetic analysis on four housekeeping genes of the isolateSGI-014-006, which are DNA gyrase subunit B (gyrB), RNA-polymerasesubunit B (rpoB), recombinase A (recA), and polyphosphate kinase (ppk).Toward this end, the entire genome of the isolate SGI-014-006 wasshot-gun sequenced, assembled and annotated by using proceduresdescribed in PCT Patent Application No. WO2010115156A2. Genomic DNA wasprepared from a fresh culture of SGI-014-006. Cell pellet was used forhigh molecular weight DNA extraction using the UltraClean® Mega Soil DNAIsolation Kit (Cat. No 12900-10) from MO BIO Laboratories, Inc.according to the manufacture's recommended protocol. The genomic DNAfrom SGI-014-006 was then prepared for shotgun 454-pyrosequencing.Genomic DNA (7.5 μg) was used for library construction according to therecommended protocol (454 Life Sciences) for single long reads. Thesequences were generated by two GS FLX Titanium series sequencing runs.

The sequences of four housekeeping genes gyrB, rpoB, recA, and ppk ofthe isolate SGI-014-006 are provided in the Sequence Listing. Homologysearch for the determined nucleotide sequences was conducted using theDDBJ/GenBank/EMBL database. Sequence identity and similarity were alsodetermined using GenomeQuest™ software (Gene-IT, Worcester Mass. USA).As discussed in details below, the result of sequence analysis of thehousekeeping genes revealed that the isolate SGI-014-006 described inthe present disclosure is a novel bacterial strain and can be consideredto be related to the family of Microbacteriaceae.

The polynucleotide sequence of the gyrB gene of SGI-014-006 has thegreatest sequence identity with a gyrB gene of Microbacterium testaceumhaving GenBank accession number AP012052 (82.69% over a 936/2172nucleotide alignment). When compared to the gyrB gene of theMicrobacterium oxydans strain DSM 20578 (GenBank AM181493; Richert etal., 2007), the sequence homologies between the two genes were ˜62%identical at the nucleotide level and ˜37% identical at the amino acidlevel.

The polynucleotide sequence of the rpoB gene of SGI-014-006 has thegreatest sequence identity with an rpoB gene of Microbacteriummaritypicum having GeneBank accession number AM181582 (96.98% over a1093/3504 nucleotide alignment). When compared to the rpoB gene of theMicrobacterium oxydans strain DSM 20578 (GenBank AM181583; Richert etal., 2007), the sequence homologies between the two genes were ˜96%identical at the nucleotide level and 99% identical at the amino acidlevel.

The polynucleotide sequence of the recA gene of SGI-014-006 has thegreatest sequence identity with a recA gene of Microbacterium testaceumhaving GeneBank accession number AP012052 (85.45% over a 962/1188nucleotide alignment). When compared to the recA gene of theMicrobacterium oxydans strain DSM 20578 (GenBank AM181527; Richert etal., 2007), the sequence homologies between the two genes were ˜92%identical at the nucleotide level and 100% identical at the amino acidlevel.

The polynucleotide sequence of the ppk gene of SGI-014-006 has thegreatest sequence identity with a ppk gene of Microbacterium luteolumhaving GenBank accession number AM181554 (91.38% over a 1380/2175nucleotide alignment). When compared to the ppk gene of theMicrobacterium oxydans strain DSM 20578 (GenBank AM181556; Richert etal., 2007), the sequence homologies between the two genes were ˜91%identical at the nucleotide level and ˜98% identical at the amino acidlevel.

Example 4: Protection of Wheat from Fusarium graminearum Infection Usingthe Microbial Antagonists Microbacterium sp. (NRRL B-50470)Mycophaerella Sp. (NRRL 50471), and Variovorax Sp. (NRRL B-50469)

Microbial strains that were found positive for antibiosis in theantagonism screen described in Example 1 were further evaluated througha plant-based bioassay in which cells of the microbial strains wereapplied directly onto seeds of a susceptible cereal cultivar, followedby inoculation with conidial spores of F. graminearum. The microbialstrains were grown to a sufficient turbidity and diluted in water. Twogram of wheat seeds of a susceptible wheat cultivar (Hank; WestBred LLC,Bozeman, Mont.) were sown in one-liter pots containing pasteurized soilmedium. After sowing, 20 mL of the microbial culture dilution wastransferred on top of sown seeds. The wheat seeds were allowed togerminate in greenhouse conditions under fluorescent lighting with a 14hour photoperiod. At the onset of wheat flowering, the wheat heads werechallenged by spraying with F. graminearum NRRL 5883 conidial spores.Conidial spores were harvested from five day old PDA plates by pouringwater with 0.01% Tween20 on plates and scraping spores into suspension.After spraying spores, wheat plants were transferred to a mist chamberwith 100% humidity for three days to allow for infection. The treatmentswere:

1. Untreated: no microbial or chemical fungicide treatment+Fusariumchallenge.

2. Non-infected: no microbial or chemical fungicide treatment, noFusarium challenge.

3. SGI-010-H11: fungal treatment+Fusarium challenge.

4. SGI-014-G01: bacterial treatment+Fusarium challenge.

5. SGI-014-006: bacterial treatment+Fusarium challenge.

Twenty days after infection, watering was stopped and wheat plants wereallowed to dry for three weeks before harvesting. Individual wheat headswere harvested and collected. Disease severity was determined for eachhead showing head blight disease symptoms. Disease severity wascalculated as the number of diseased spikelets divided by the totalnumber of spikelets per heads. The results (TABLE 2) revealed that wheatplants treated with each of the three microbial antagonists tested,SGI-014-006 (NRRL B-50470), SGI-010-H11 (NRRL 50471), and SGI-014-G01(NRRL B-50469), had significantly lower severity and incidence ofFusarium graminearum infestation when compared to the “untreated”control growing in the same conditions (P<0.05).

The seeds from each head were harvested by hand and the seed mass wasweighed. The average seed weight per pot in each of the treatments wascalculated. As reported in TABLE 3, each of the microbial antagonistswas found to significantly reduce yield loss caused by head blightinfestation.

TABLE 2 Efficacy of the microbial antagonists in reducing head blightincidence in wheat. Treatment Percent severity (%) STDEV P valueNon-infected  1.30 0.0025 <.0001 Untreated 15.46 0.0364 NA SGI-010-H11 7.42 0.0219  0.0005 SGI-014-G01  6.59 0.0315  0.0001 SGI-014-C06  7.210.0225  0.0004

TABLE 3 Efficacy of the microbial antagonists in preserving seed yieldin wheat plants having head blight disease. Treatment Seed mass (g) Pvalue Non-infected 0.8438 ± 0.1633 0.0191 Untreated 0.4669 ± 0.1209 NASGI-010-H11 0.6200 ± 0.1844 0.7025 SGI-014-G01 0.7963 ± 0.2032 0.5277SGI-014-C06 0.6744 ± 0.0948 0.0551

Example 5: Growth Inhibition of Fusarium graminearum by MicrobialAntagonists on Wheat Seedlings

The microbial strains found to possess suppressive activity against F.graminearum in antagonism plate assay were investigated further fortheir ability to reduce the incidence of head blight in wheat. Seeds ofa susceptible wheat cultivar (Hank; WestBred LLC, Bozeman, Mont.) weresown in 1-Liter pots each containing pasteurized soil medium in 20 cmdiameter plastic pots. Microbial cell suspensions were prepared asfollows.2YT, or similar growth medium, broth cultures were inoculatedfrom isolate glycerol stocks or streak plates. Typically, cultures wereinitiated 48-72 hours prior to use in the growth chamber, greenhouse orfield allowing the culture to reach late exponential phase. Isolatesthat have longer doubling times were initiated further in advance.Cultures were incubated at 30° C. on a rotary shaker at 200 rpm. Aftergrowth, the cells were pelleted at 10,000×g for 15 min at 4° C. andre-suspended in 10 mM MgSO₄ buffer (pH 7.0). Cell densities werenormalized for each isolate on a CFU/mL basis (cell performing units permilliliter). Typically, ˜10⁹ CFU/mL suspensions were prepared for eachisolate and transported to the inoculation site on ice. Inoculationswere performed by diluting these cell suspensions 1/20 in irrigationwater to a final density 5×10⁷ CFU/mL. For 1-L pot trials, 20 mL of thisdilute cell suspension was distributed evenly over the surface of eachreplicate pot. For micro-trial pots, cell suspensions were not dilutedand 1 mL of each 10⁹ CFU/mL suspension was pipetted directly onto thesurface of each replicate pot as soil drench on top of the submergedseeds. The seeds and plants were maintained in a greenhouse at roomtemperature with a 14 h photoperiod of light.

Mycelium of the Fusarium graminearum strain NRRL-5883 was grown on PDAplates for 5 days under constant light. Hyphae and conidia wereharvested by pouring a few mL of sterile water (0.01% Tween 20) on theplates and scraping the agar surface with a sterile spatula. Theconcentration of spores in the inoculum was approximately 2×10⁵spores/mL, but hyphal fragment concentration was not determined.

Inoculation with F. graminearum began after shoot emerging from theseeds and continued every other evening for 10 days. The inoculum of F.graminearum was applied with a sprayer at about 30 mL per pot.Immediately after each inoculation, the plants were misted with overheadmister. When chemical fungicide was used, the fungicide (Banner Maxx,Syngenta) was prepared by diluting 2 mL of fungicide stock in 1 L ofdistilled water, and was applied with a sprayer at about 30 mL per pot.

Head blight was evaluated by the severity of Fusarium infestation, asdetermined by the number of Fusarium colony-forming units per gram(CFU/g) of plant tissues. All treatments were performed in four blockswith four replicate pots. The treatments were:

1. Untreated: no microbial or chemical fungicide treatment, +Fusariumchallenge

2. Non-infected: no microbial or chemical fungicide treatment, noFusarium challenge.

3. Fungicide: chemical fungicide treatment+Fusarium challenge.

4. SGI-010-H11: fungal treatment+Fusarium challenge.

5. SGI-014-G01: bacterial treatment+Fusarium challenge.

6. SGI-014-006: bacterial treatment+Fusarium challenge.

The results, as reported in TABLE 4, revealed that all three microbialtreatments provided a significant decrease in FHB severity as comparedto the infected control. In particular, two microbial antagonists,SGI-014-006 and SGI-010-H11, significantly reduced the infestation ofwheat plants by Fusarium graminearum when compared to the untreatedcontrol grown in the same conditions. The protection of wheat plantsfrom Fusarium infestation effected by each of the two microorganismsSGI-014-006 and SGI-010-H11 was statistically comparable to theprotection effected by the commercial chemical fungicide. When themicroorganism SGI-014-G01 was applied, the observed protection of wheatplants from Fusarium infestation was much less conspicuous and itseffectiveness varied greatly among the replicate pots.

TABLE 4 Effect of microbial antagonists on Fusarium infestation.Treatment CFU/g P value Untreated 1487 ± 600 N/A Non-infected   0 ± 0<.0001 Chemical fungicide  290 ± 146 <.0001 SGI-010-H11  671 ± 450 0.0077 SGI-014-G01 1246 ± 465  0.8387 SGI-014-C06  367 ± 234  0.0001

Example 6: Growth and Storage of the Microbial Antagonists

Mycosphaerella sp.: Several methods were used to store the isolatedfungus as a pure culture, one of which was the filter paper technique.The fungus was also allowed to grow on PDA, and then it was cut intosmall squares which were placed into vials containing 15% glycerol andstored at −70° C. The fungus was also stored at 4° C. by a similarmethod, using distilled water rather than glycerol. However, one of thepreferred methods of storage was on infested sterile barley seed at −80°C.

Bacillus sp., Microbacterium sp. and Variovorax sp.: The isolatedbacteria were stored as a pure culture. A bacterial colony wastransferred to a vial containing R2A broth liquid medium (Tecknova) andallowed to grow at 30° C. with shaking at 250 rpm for two days. Theculture was then transferred into vials containing 15% glycerol andstored at −80° C.

Example 7: Spore Production and Seed Coating Treatments

Spore production: In a typical spore production procedure, one liter of2×YT growth medium (16 g/L Tryptone, 10 g/L yeast extract, 5 g/L NaCl)was inoculated with 5 mL starter culture or petri dish scraping andincubated overnight at 30° C. in a rotating shaker that was set at 225rpm. Bacterial cells were pelleted by centrifugation, and washed 1× withPBS buffer (8 g/L NaCl; 0.2 g/L KCl; 1.44 g/L Na₂HPO₄; 0.24 g of KH₂PO₄;pH 7.4). Cells were resuspended in CDSM medium (Hageman et al., J.Bacteriol., 438-441, 1984), and were grown for four additional nights at30° C. in the rotating shaker. Sporulation in the bacterial culture wasmonitored daily by using phase contrast microscope until the entireculture was virtually made up of free-floating spores. The incubationtime typically took less than four days or more than six days dependingon the species. Under phase contrast microscope, endospores weredetected within cells as bright white oblate spheroids. Bacterial sporeswere pelleted by centrifugation at 10,000×g for 15 minutes, washed twotimes with sterile dH₂0 and, if necessary, concentrated down to 50 mLand could either be used immediately or refrigerated for later use.Spore concentration was measured at OD₆₀₀. This procedure typicallyproduced at least 2000 OD's of bacterial spores.

In particular, several bacterial strains of the present invention, e.g.Bacillus sp. SGI-015-F03, could conveniently produce large amounts ofspores after 4 days of incubation with a simple inoculation of a largeovernight starter culture (—15 mL) in to 1 liter of 2× SG growth medium,followed by a 4-day incubation at appropriate temperature in a rotatingshaker set at 225 rpm. The recipe of the 2× SG growth medium was asfollows: 16 g/L Nutrient Broth; 0.25 g/L MgSO₄; 2.0 g/L KCl; 0.15 g/LCaCl₂.2H₂O; 0.025 g/L MnCl₂.2H₂O; 0.28 mg/L FeSO₄.7H₂O; 1.0 g/LDextrose.

Seed coating treatments of wheat and corn seeds: Small-scale seedtreatment experiments were conducted by following a procedure describedin Sudisha et al., 2009 with minor modifications. Typically, abiopolymer stock solution was made by adding 6 gram of gum Arabic powder(MP Biomedical) to 36 mL water in a 50 mL Falcon tube, which wassubsequently mixed to homogeneity by using a wheel mixer. A stir platewas used for mixing when larger quantities of coated seeds were needed.

When vegetative cells were used, turbid cultures of actively growingmicrobial cells were washed with PBS and adjusted to an OD₆₀₀ of ˜5.0.Alternatively, microbial spore suspensions were prepared as describedabove. Bacterial spores and/or vegetative cells were thoroughlyresuspended in ˜32 mL gum Arabic biopolymer stock solution prepared asdescribed above, and resulting suspension was mixed thoroughly in a 1 Lbottle. Approximately 400 g of seeds (either wheat or corn) were addedto the bottle and vigorously shaken or vortexed to ensure a uniformdistribution of the gum/cell suspension. Coated seeds were then spreadacross a sterile plastic weigh boats to dry in a laminar flow hood untilno longer tacky, generally 3-6 hours with periodic mixing. In someinstances, seeds coated with spores could be dried overnight. However,seeds coated with vegetative cultures were typically stored away beforethey completely desiccated. Viability test performed periodically on themicrobes used in seed coating formulation showed that the microbesremained viable for at least three months. Germination rate of thecoated seeds was determined to be essentially identical to controluncoated seeds.

Example 8: Effect of Microbial Seed Treatments on the Development ofFusarium Head Blight in Wheat

Seeds of a FHB-susceptible wheat cultivar (RB07) were coated with eachof the microorganisms SGI-014-006; SGI-015-F03, and SGI-015-H06, inaccordance with the procedure described in Example 8. Coated seeds weresubsequently sown in one-liter pots containing pasteurized soil medium[(Metromix-Mix 200; Scotts-Sierra Horticultural Products, Marysville,Ohio; and 3 gram of fertilizer 14-14-14 (N—P—K)]. Pots were seeded at7-8 plants per pot and subsequently thinned to 5 plants per pot at the3-leaf stage. Pots were arranged in randomized blocks with 5 pots pertreatments. The coated seeds were then allowed to germinate ingreenhouse conditions under fluorescent lighting with 16 hours oflighting per day. At the onset of wheat flowering (anthesis), the wheatheads were challenged by spraying with F. graminearum NRRL 5883macroconidial spores at a concentration of 100,000 spores/mL. Afterspraying spores, wheat plants were transferred to a dew chamber with100% humidity for three days to allow for infection. The treatmentswere:

1. Untreated: no microbial or chemical fungicide treatment+Fusariumchallenge.

2. SGI-014-006: bacterial seed treatment+Fusarium challenge.

3. SGI-015-F03: bacterial seed treatment+Fusarium challenge.

4. SGI-015-H06: bacterial seed treatment+Fusarium challenge.

Severity of head blight disease was assessed visually at ten days andtwenty-one days after infection. Disease severity was determined foreach spike showing head blight disease symptoms, and calculated as thepercentage of symptomatic spikelets; i.e. the number of spikeletsdisplaying FHB symptoms divided by the total number of spikelets. Theresults (TABLE 5) revealed that wheat seeds coated with each of thethree microbial antagonists tested, SGI-014-006; SGI-015-F03, andSGI-015-H06, had significantly lower severity of Fusarium graminearuminfestation when compared to the “untreated” control growing in the sameconditions (P<0.05).

TABLE 5 Development of head blight symptoms in wheat following seedtreatments with antagonistic microorganisms Infection severity (%) 10days 21 days Treatment post-infection post infection Untreated control49.31 66.10 SGI-014-C06  2.08  7.74 SGI-015-F03 23.12 31.53 SGI-015-H06 9.42 19.81

Example 9: Biocontrol of Head-Blight Disease of Wheat in GreenhouseTrials

Each of the microorganisms SGI-014-006; SGI-015-F03, and SGI-015-H06were further tested at larger-scale greenhouse trials. The trials wereconducted in a “plant growth containment room” (PGCR) located atSynthetic Genomics, Inc., and included the following control treatments:infected control, non-infected control, as well as four commercialfungicide benchmarks. The benchmarking chemical fungicides were BannerMAXX® (Syngenta) at a concentration of 2 ml/L and Prosaro® (BayerCropScience) at a concentration of 3 ml/L, each applied as a foliarspray, following manufacturers' recommendation. The benchmarkingbiological fungicides were Actinovate® (Natural Industries) andRhizoVital® (ABiTEP GmbH), each applied as a soil drench as perinstruction manuals recommended by manufacturers.

The microbial treatments were prepared and applied either as a soildrench or as a seed coat. When microbial treatments were applied as asoil drench, cell suspensions of each of the microorganisms wereindividually applied to the seeds before covering the seeds with moregrowth medium. For this purpose, freshly prepared cultures were pelletedto remove growth media and resuspended in 10 mM magnesium sulfate(MgSO₄) buffer. Cell suspensions were then added by pipetting anddistributing 20 milliliters evenly across the seeds at a total cellnumber of ˜10⁹ cells per pot. When microbial treatments were applied asa seed coating, microorganism cells/spores were essentially prepared asdescribed in Example 8 above. Coated seeds were produced byincorporating cell suspensions with a gum Arabic solution to form atacky coating mixture that was subsequently applied to the seeds. Themicrobial seed coating was then allowed to dry to form a hard but watersoluble biopolymer entrapping the microorganism cells/spores. The goalwas to reach a cell titer in the range of at least 10⁶-10⁷ viablecells/seed. In this greenhouse trial experiment, the seed coatings wereprepared the day prior to the sowing of the seeds.

Each treatment had four replicates that were positioned in a RandomizedComplete Block Design (RCBD) on three leveled racks arranged in two rowsalong the sides of a growth room. The racks were equipped withfluorescent lights (Agrosun, 5850K) that generated on average 800 moleof light intensity as measured at the pot surface. Each replicateconsisted of four 1-liter pots contained in a half flat. The pots werefilled with a synthetic growth medium consisted of a 3:1 ratio (v/v) ofArabidopsis Growth Medium AIS (Lehle Seeds) to sand (Washed Coarse). Ineach pot, two grams of spring wheat seeds (Hank, WestBred) werescattered on the surface of the growth medium. The pots were bottomwatered by keeping ˜2 cm water at their base and observed forgermination and growth.

At flowering stage (Feekes 10.5.1) the wheat heads were infected byspraying with a Fusarium graminearum conidial spore suspension. For thisgreenhouse trial experiment, fungal spores were prepared by platinghomogenized mycelia of F. graminearum strain NRRL 5883 onto large PDAplates, followed by five-day incubation under constant light at roomtemperature. The fungal spores were then harvested by flooding theplates with magnesium sulfate buffer (10 mM MgSO₄; 0.01% Tween 20) andgently scraping the agar surface with a sterile spatula. The sporeconcentration was adjusted and approximately 50,000 conidial spores wereapplied per head with a pressurized hand sprayer. The growth roomrelative humidity was increased to 90% for three days allowing forinfection before again being normalized. After wheat seeds had set,watering was stopped and the wheat heads were allowed to dry completely.Individual wheat heads were harvested and collected. Disease severitywas determined for each wheat head showing head blight disease symptoms.Disease severity was calculated as the number of diseased spikeletsdivided by the total number of spikelets per head. The results (TABLE 6)revealed that wheat treated with each of the three microbial antagoniststested, SGI-014-006; SGI-015-F03, and SGI-015-1106, had significantlylower severity and incidence of Fusarium graminearum infestation whencompared to the “untreated” control growing in the same conditions(P<0.05).

TABLE 6 Efficacy of the microbial antagonists in reducing head blightincidence in wheat. Infection Infection Treatment severity (%)protection % Non-infected  2.1 ± 2.9 N/A Infected 37.2 ± 9.8  0.00Prosario ®  4.6 ± 2.8 93.00 Banner-MAXX ® 12.5 ± 5.8 70.00 Actinovate ®38.9 ± 12.1 −5.00 RhizoVital ® 28.0 ± 5.4 26.00 SGI-014-C06 18.4 ± 9.130.00 SGI-015-F03 25.4 ± 6.9 54.00 SGI-015-H06 26.6 ± 7.4 34.00

To determine seed yield, wheat heads were individually threshed by hand,the seeds collected and cleaned of chaff and other detritus, counted,and weighed. The total seed yield was determined as the total seed massproduced by 16 pots of each of the treatments. As reported in TABLE 7each of the three microbial antagonists tested was found tosignificantly protect the seed yield; i.e. the yield loss caused by headblight infestation was significantly reduced.

TABLE 7 Efficacy of the microbial antagonists in preserving seed yieldin wheat plants having head blight disease. Total seed Yield Treatmentyield (g) protection (%) Non-infected 14.36 N/A Untreated 11.05  0.00Prosario ® 13.44  72.00 Banner-MAXX ® 14.41 102.00 Actinovate ® 11.99 28.00 Rhizo Vital ® 12.65  48.00 SGI-014-C06 14.70 110.00 SGI-015-F0315.03 120.00 SGI-015-H06 14.14  93.00

Thus, treatments of wheat plants with each of the tested microorganismssignificantly preserved the wheat plants against yield loss caused byFusarium infestation. In particular, wheat plants treated with eitherSGI-014-006 or SGI-015-F03 showed a significant improvement in totalseed yield; i.e. 110% and 120% respectively, when compared tonon-infected control. In contrast, the benchmarking fungicidesActinovate®, RhizoVital®, and Prosario® did not appear to significantlyprotect the treated wheat plants from yield loss caused by Fusarium headblight infestation.

Example 10: Biocontrol of Wheat Scab Under Field Conditions

Microorganism antagonists that were found having suppressive activityagainst the pathogen F. graminearum and head blight disease in in vitroantagonism assays and greenhouse studies, as described in Examples 4-9,are further evaluated in a series field experiments at differentgeographical locations throughout the United States. Some experimentsare carried out in agricultural areas which are used for microbiologicalcontrol of wheat scab, where natural disease infection occurs. The wheatvariety used in the test is a F. graminearum susceptible variety.Generally, wheat plants with each treatment were sown in plots of 12rows, about 3-meters long. The space between rows is about 20 cm.Treated plots in each experiment are arranged in a randomized blockdesign. The efficacy of various wettable powder compositions containingthe microbial antagonists of the present invention in reducing headblight severity and incidence are also assessed. In these experiments,the microbial antagonists are evaluated either individually or incombination.

In these tests, microorganism antagonists are evaluated in variousseed-coating treatments and/or field tests where microbial suspensionsare applied directly onto flowering wheat heads. In each of theexperiments, the microorganisms are assessed in replicated plots.Antagonist, pathogen, and plant production methods as described inExamples 4-9 can be used in these experiments.

Seed-coating treatment—In addition to the seed coating method describedin Example 7 above, a variety of techniques and seed-coatingformulations known in the art can be deployed for the treatment of wheatseeds with microbial antagonists, such as those described previously byFernando et al., 2002; Bello et al., 2002; and Kim et al., 1997. Ingeneral, wheat seeds are pre-moistened and stored at room temperature topromote germination. Germinated seeds can be then immersed intomicrobial suspensions before seeding. The pathogen spores can beinoculated either before or after bacterial inoculation. Antagonist,pathogen, and plant production methods as described in Examples 4 and 5can be used for these seed treatments.

Disease severity and yield assessment—The antagonists are subjected tofurther evaluation in greenhouse and field evaluation for theirsuppressive activity against head blight disease on wheat and barley atflowering stages, by using a variety of methods and procedures,typically those described in, e.g., Schisler, Plant Disease, Vol 86(12),1350-1356, 2002; Schisler et al. Biological Control, 39:497-506, 2006;and Khan and Doohan, Biological Control, 48:42-47, 2009. In one of suchexperiments, inoculums of G. zeae are prepared on sterile, yellow dentcorn as described by Khan et al. (Biological Control, 29:245-255, 2004).Fully colonized lipid cultures (48-hour culture) of microbial strainsare diluted to one-quarter strength with phosphate buffer. Final CFU permL counts for antagonist treatments are between 1×10⁹ CFU/mL and 6×10⁹CFU/mL. Treatment suspensions are then applied to flowering wheat headsusing a CO₂ backpack sprayer. Treatments are typically applied aftersunset to minimize potential UV degradation of antagonist cells. Thereare 5 replicates per treatment which are arranged in a randomized blockdesign. The primary control treatment consists of plants treated with abuffer solution. A second control consists of untreated plants. Fieldassessments of head blight disease and incidence are made by evaluating60 heads per replicate (L e. 300 heads/treatment) when plant developmentreaches between mid-milk and soft dough development. Wheat heads areharvested by hand and threshed using an Almaco single plant and headthresher (Almaco, Iowa) when grains reach full maturity. Grain samplesobtained from each replicate row are evaluated for 100-kernel weight.Disease severity, incidence, and 100-kernel weight data are analyzedusing one-way analysis of variance (ANOVA).

The compositions containing the microbial antagonists of the invention,either individually or in combination, are found to significantlydiminish the disease incidence. These results show that the biologicalcontrol measures using these active microbial agents can play animportant role in the management of scab on cereal plants such as wheatplants.

Example 11: Development of Non-Naturally Occurring Cultivars andBreeding Program

Endophytic microorganisms of the present invention are introduced intocrop plants, including cereals, of varying genotypes and geographicorigin, lacking such endophytic microorganisms, to createplant-endophyte combinations with improved agronomic characteristics,using procedures analogous to those known in the art, including thosedescribed in U.S. Pat. Appl. No. 20030195117A1; U.S. Pat. Appl. No.20010032343A1; and U.S. Pat. No. 7,084,331, among others. Thus, givensynthetic plant-endophyte combinations may be created and selected in abreeding/cultivar development program based on their ability to form andmaintain a mutualistic combination that results in an agronomic benefit.Rating of agronomic characteristics of the combination may also beutilized in such a breeding program. These characteristics may include,without limitation, drought tolerance, biomass accumulation, resistanceto insect infestation, palatability to livestock (e.g. herbivores), easeof reproduction, and seed yield, among others. Such combinations maydiffer in levels of accumulation of microbial metabolites that are toxicto pests and weeds, including ergot alkaloid levels, loline levels,peramine levels, or lolitrem levels, while displaying desired agronomiccharacteristics of crop plants, including resistance to insect feedingor infestation, resistance to abiotic stress, palatability to livestock,biomass accumulation, ease of reproduction, and seed yield, among othertraits.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that elements of the embodimentsdescribed herein can be combined to make additional embodiments andvarious modifications may be made without departing from the spirit andscope of the invention. Accordingly, other embodiments, alternatives andequivalents are within the scope of the invention as described andclaimed herein.

Headings within the application are solely for the convenience of thereader, and do not limit in any way the scope of the invention or itsembodiments.

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically canindividually indicated to be incorporated by reference.

1. A microbial strain, wherein the microbial strain is a Variovorax sp.strain, and wherein said microbial strain has suppressive activityagainst head blight disease.
 2. The microbial strain according to claim1, wherein said microbial strain is Variovorax sp. strain SGI-014-G01(deposited as NRRL B-50469) or a pesticidally active variants thereof.3. The microbial strain according to claim 2, wherein said microbialstrain comprises a DNA sequence exhibiting at least 95% sequenceidentity to SEQ ID NO:12. 4-5. (canceled)
 6. A composition comprisingthe microbial strain of claim 1 or a culture thereof, and anagriculturally effective amount of a compound or composition selectedfrom the group consisting of an acaricide, a bactericide, a fungicide,an insecticide, a microbicide, a nematicide, a pesticide, and afertilizer.
 7. A composition comprising the microbial strain of claim 1or a culture thereof, and a carrier.
 8. A composition according to claim7, wherein said carrier is an agriculturally acceptable carrier.
 9. Acomposition according to claim 7, wherein said carrier is a plant seed.10. A composition according to claim 7, wherein said composition isprepared as a formulation selected from the group consisting of anemulsion, a colloid, a dust, a granule, a pellet, a powder, a spray, anemulsion, and a solution.
 11. A composition according to claim 7,wherein said composition is a seed coating formulation.
 12. A seedhaving a coating comprising a composition according to claim
 7. 13. Amethod for preventing, inhibiting or treating the development of a plantpathogen, wherein said method comprises growing the microbial strain ofclaim 1 or a culture thereof in a growth medium or soil of a host plantprior to or concurrent with host plant growth in said growth medium orsoil.
 14. A method according to claim 13, wherein said plant pathogencauses head blight disease.
 15. A method according to claim 14, whereinsaid plant pathogen is Fusarium graminearum.
 16. A method forpreventing, inhibiting or treating the development of head blightdisease of a plant, wherein said method comprises applying to the plant,or to the plant's surroundings, an effective amount of the microbialstrain of claim 1 or a culture thereof.
 17. A method according to claim16, wherein said microbial strain or culture thereof is applied to soil,a seed, a root, a flower, a leaf, a portion of the plant, or the wholeplant.
 18. (canceled)
 19. A method according to claim 16, wherein saidplant is a wheat plant, a corn plant, a barley plant, or an oat plantand/or wherein said plant is susceptible to Fusarium graminearum.
 20. Amethod according to claim 16, wherein said microbial strain or culturethereof is established as an endophyte on said plant.
 21. Anon-naturally occurring plant that is a plant artificially infected withthe microbial strain of claim 1 or a culture thereof.
 22. Seed,reproductive tissue, vegetative tissue, plant parts, or progeny of anon-naturally occurring plant of claim
 21. 23. A method for preparing anagricultural composition, wherein said method comprises inoculating themicrobial strain of claim 1 or a culture thereof into or onto asubstratum and allowing said microbial strain or culture thereof to growat a temperature of 1-37° C. until obtaining a number of cells or sporesof at least 10²-10³ per milliliter or per gram.